PRKD1 Antibody, HRP conjugated

What is PRKD1 and what are its key functions in cellular processes?

PRKD1, also known as Protein Kinase D1 (PKD1), functions as a serine/threonine protein kinase (EC 2.7.11.13) involved in multiple cellular processes. It is alternatively known as Protein Kinase C mu type (PRKCM), nPKC-D1, or nPKC-mu . PRKD1 plays crucial roles in signal transduction pathways, particularly those affecting cancer progression and metastasis . Research has shown that PRKD1 functions as an upstream regulatory kinase of metastasis-associated protein 1 (MTA1), directly impacting cancer metastatic activity . In colon cancer, PRKD1 has been shown to attenuate tumorigenesis by modulating the β-catenin/TCF-4 transcription complex formation . In breast cancer, PRKD1 expression is frequently silenced through epigenetic mechanisms, with its restoration inhibiting invasion and metastasis .

How should PRKD1 antibodies be stored to maintain optimal activity?

PRKD1 antibodies, including HRP-conjugated variants, require specific storage conditions to maintain their reactivity and specificity. The general recommendation is to store these antibodies at -20°C upon receipt . For long-term storage, some manufacturers suggest -80°C as an alternative . It is crucial to avoid repeated freeze-thaw cycles as this can degrade antibody quality and compromise experimental results . Most commercial preparations are supplied in a storage buffer containing glycerol (typically 50%), which prevents freezing at -20°C and helps maintain antibody stability . For instance, the PRKD1 antibody available from Qtonics (QA55732) is supplied in a buffer containing 50% glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative .

What is the typical molecular weight of PRKD1 protein when detected by antibodies?

The calculated molecular weight of PRKD1 protein is approximately 102 kDa as reported in product information sheets . This information is essential for accurate band identification when performing Western blot analysis. When designing experiments to detect PRKD1, researchers should anticipate visualization of protein bands around this molecular weight, keeping in mind that post-translational modifications may slightly alter the apparent molecular weight on SDS-PAGE gels. The GenBank accession number for human PRKD1 is NM_002742, and the gene ID (NCBI) is 5587, which can be useful for verifying sequence information when troubleshooting antibody specificity issues .

How can PRKD1 antibodies be utilized to study protein-protein interactions in cancer pathways?

PRKD1 antibodies can be instrumental in studying protein-protein interactions through co-immunoprecipitation (co-IP) experiments and proximity ligation assays (PLA). Research by Nature has demonstrated that PRKD1 physically interacts with MTA1, and this interaction is critical for regulating cancer metastasis . To investigate such interactions, investigators have successfully employed immunoprecipitation techniques followed by Western blotting. For example, in studies of colon cancer, equal amounts of nuclear lysate proteins from SW480-PKD1-GFP or SW480-GFP cells were immunoprecipitated using anti-TCF-4 antibody, and the resulting complexes were analyzed by Western blotting with antibodies against β-catenin, PKD1, and TCF-4 . This approach revealed that PRKD1 overexpression led to decreased β-catenin/TCF-4 transcription complex formation . For advanced interaction studies, proximity ligation assays can provide spatial resolution of PRKD1 interactions within intact cells, offering advantages over traditional co-IP methods.

What are the optimal protocols for detecting phosphorylated forms of PRKD1?

Detection of phosphorylated PRKD1 requires phospho-specific antibodies and careful sample preparation to preserve phosphorylation status. While the provided search results don't specifically address phospho-PRKD1 detection, the methodological approach would include:

• Rapid cell lysis in the presence of phosphatase inhibitors

• Sample preparation under denaturing conditions to maintain phosphorylation status

• Use of phospho-specific PRKD1 antibodies targeting key regulatory sites

• Validation of specificity using phosphatase treatment controls

For cell-based assays, researchers can use immunofluorescence to detect spatiotemporal changes in PRKD1 phosphorylation. The recommended dilution for immunofluorescence applications with PRKD1 antibodies is 1:50-1:500, though this should be optimized for each experimental system . For flow cytometry detection of intracellular phospho-PRKD1, approximately 0.25 μg antibody per 10^6 cells in a 100 μl suspension is recommended .

How can PRKD1 antibodies be used to study epigenetic silencing in cancer progression?

PRKD1 expression is frequently silenced through epigenetic mechanisms in invasive cancers, making it an excellent model for studying epigenetic regulation. Research published in PMC demonstrated that the PRKD1 promoter is aberrantly methylated in invasive breast cancer cells, with methylation increasing with tumor aggressiveness . To study such epigenetic modifications, researchers employed several complementary techniques:

• Reduced representation bisulfite deep sequencing to analyze PRKD1 promoter methylation

• Methylation-specific PCR (MSP-PCR) and in situ MSP-PCR

• Reexpression strategies using DNA methyltransferase inhibitors like decitabine

• Quantification of PRKD1 gene and exon expression levels using PCR with primers designed for template regions recommended by SnowShoes-FTD

Using these methods, investigators demonstrated that reversion of PRKD1 promoter methylation with decitabine restored PKD1 expression and blocked tumor spread and metastasis in a PKD1-dependent manner . For analyzing PRKD1 mRNA expression, PCR reactions were typically carried out under conditions of 1 minute at 55°C followed by a 1-minute extension at 72°C for 35 cycles .

What controls should be included when using PRKD1 antibodies in Western blot experiments?

When conducting Western blot experiments with PRKD1 antibodies, several essential controls should be incorporated:

• Positive Control: Cell lysates from cells known to express PRKD1, such as HeLa or LNCaP cells, should be included

• Negative Control: Either PRKD1 knockout cell lines or cell lines with confirmed low PRKD1 expression

• Loading Control: Antibodies against housekeeping proteins like GAPDH (primers: 5′-TCAACGGATTTGGTCGTATTG-3′ and 5′-AGAGTTAAAAGCAGCCCTGGTGA-3′)

• Peptide Competition: Pre-incubation of the antibody with immunizing peptide to confirm specificity

• Molecular Weight Marker: To confirm the detected band is at the expected molecular weight (102 kDa for PRKD1)

For HRP-conjugated PRKD1 antibodies, an additional control omitting the primary antibody should be included to assess potential non-specific binding of detection reagents.

What are the recommended dilutions for different applications of PRKD1 antibodies?

Optimal dilutions vary depending on the specific application and should be empirically determined for each experimental system. Based on manufacturer recommendations, the following dilution ranges serve as starting points:

As noted in product literature: "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" and results may be "sample-dependent" .

How can researchers troubleshoot inconsistent results when using PRKD1 antibodies in different cell lines?

Inconsistent results across cell lines may stem from several factors that require systematic troubleshooting:

• Variable PRKD1 Expression Levels: Expression levels can differ dramatically between cell lines. For accurate comparisons, quantify baseline PRKD1 expression using qRT-PCR with primers specific for PRKD1 .

• Post-translational Modifications: PRKD1 undergoes phosphorylation and other modifications that may affect antibody recognition. Ensure consistent sample preparation across cell lines.

• Splice Variants: Different cell lines may express different PRKD1 isoforms. Verify which domain your antibody targets (e.g., the Qtonics antibody targets residues 674-904AA of human PRKD1) .

• Subcellular Localization Differences: PRKD1 can shuttle between nuclear and cytoplasmic compartments . Consider separate analysis of nuclear and cytoplasmic fractions.

• Epigenetic Silencing: The PRKD1 promoter is frequently methylated in cancer cells . Treatment with demethylating agents may be necessary to restore expression in some cell lines.

When comparing results across cell lines, normalize loading carefully and consider using multiple antibodies targeting different PRKD1 epitopes to validate findings.

How can researchers utilize PRKD1 antibodies to study its role in metastasis suppression?

PRKD1 has been identified as a significant metastasis suppressor, making it a valuable target for cancer research. A comprehensive methodological approach to studying its role in metastasis would include:

• In Vitro Migration/Invasion Assays: Transwell invasion assays with cells manipulated to overexpress or knock down PRKD1 have been effectively used to study its anti-invasive properties .

• Protein-Protein Interaction Studies: Co-immunoprecipitation and proximity ligation assays (PLA) have demonstrated that PRKD1 physically interacts with metastasis-associated protein 1 (MTA1) . These techniques can reveal the mechanistic basis of PRKD1's metastasis-suppressive functions.

• Animal Models: Several animal models have proven valuable, including:

  • Intra-tibial xenograft models

  • Subcutaneous xenograft models

  • PTEN-knockout (PTEN-KO) mice

  • Transgenic adenocarcinoma of mouse prostate (TRAMP) models

• In Vivo Imaging: The IVIS Spectrum Pre-clinical In Vivo Imaging System has been used to monitor tumor growth and metastasis in real-time in animal models following manipulation of PRKD1 expression .

• Clinical Correlation Studies: Correlation between PRKD1 and metastatic markers in human cancer tissues can provide translational relevance to experimental findings .

Research has shown that PRKD1 regulates metastasis by phosphorylating MTA1, supporting its nucleus-to-cytoplasmic redistribution, and promoting its polyubiquitin-dependent proteosomal degradation .

What are the best approaches for analyzing PRKD1 expression in clinical tissue samples?

Analysis of PRKD1 expression in clinical samples requires careful consideration of tissue heterogeneity and preservation of protein integrity. Recommended methodological approaches include:

• Immunohistochemistry (IHC): PRKD1 antibodies can be used for IHC analysis of formalin-fixed, paraffin-embedded (FFPE) tissue sections. Both polyclonal antibodies, like the rabbit polyclonal antibody from Qtonics , and monoclonal antibodies can be employed, though each requires optimization of antigen retrieval and detection methods.

• Methylation Analysis: Since PRKD1 is frequently silenced by promoter methylation, methylation-specific PCR (MSP-PCR) and in situ MSP-PCR provide valuable insights into the epigenetic regulation of PRKD1 in clinical samples . Studies have successfully employed these techniques across a spectrum of breast cancer subtypes, including:

  • 34 cases of "normal" tissue

  • 22 cases of ductal carcinoma in situ

  • 22 cases of ER+/HER2- invasive lobular carcinoma

  • 43 cases of ER+/HER2- invasive ductal carcinoma

  • 93 cases of HER2+ invasive ductal carcinoma

  • 96 cases of triple-negative invasive ductal carcinoma

• RNA Expression Analysis: RT-PCR and quantitative real-time PCR can assess PRKD1 mRNA levels in fresh or frozen tissue samples. Expression levels can be normalized using housekeeping genes like GAPDH .

• Protein Extraction and Western Blotting: For quantitative protein analysis, Western blotting using PRKD1-specific antibodies can be performed on protein extracts from clinical samples.

How can researchers design experiments to study the subcellular localization and trafficking of PRKD1?

Understanding PRKD1's subcellular localization is critical as it shuttles between different cellular compartments to exert its functions. Experimental approaches to study this dynamic process include:

• Immunofluorescence Microscopy: The recommended dilution for immunofluorescence applications with PRKD1 antibodies is 1:50-1:500 . This technique has been validated in HeLa and LNCaP cells and can reveal the spatial distribution of PRKD1 within intact cells.

• Subcellular Fractionation: Biochemical separation of nuclear, cytoplasmic, and membrane fractions followed by Western blotting can quantify PRKD1 distribution across cellular compartments. This approach was used to demonstrate that PRKD1 overexpression decreased nuclear β-catenin/TCF-4 complex formation in colon cancer cells .

• Live-Cell Imaging: Fusion of PRKD1 with fluorescent tags like GFP enables real-time tracking of its movement between cellular compartments in response to various stimuli. SW480-PKD1-GFP cells have been successfully used in such experiments .

• Protein Transport Inhibition Studies: Treatment with specific inhibitors of nuclear import/export can help elucidate the mechanisms regulating PRKD1 trafficking.

• Co-localization Studies: Dual immunofluorescence staining for PRKD1 and known interacting partners like MTA1 can reveal functional associations in different subcellular compartments .

Research has shown that PRKD1 facilitates the nuclear-to-cytoplasmic redistribution of MTA1, which is a critical step in reducing MTA1's oncogenic activity .

How do different fixation methods affect PRKD1 antibody performance in immunofluorescence studies?

Fixation protocols can significantly impact the performance of PRKD1 antibodies in immunofluorescence applications. While the search results don't provide specific details on fixation optimization, general considerations for PRKD1 immunofluorescence include:

• Paraformaldehyde Fixation: 4% paraformaldehyde (PFA) is commonly used for preserving cellular architecture while maintaining protein antigenicity. For PRKD1 detection, a fixation time of 10-15 minutes at room temperature is typically sufficient.

• Methanol Fixation: Cold methanol fixation may enhance detection of certain epitopes by exposing antigenic sites, but can disrupt membrane structures. This may be particularly relevant for PRKD1, which interacts with membrane lipids.

• Permeabilization: When using PFA fixation, subsequent permeabilization with 0.1-0.5% Triton X-100 or 0.05% saponin is necessary for antibody access to intracellular PRKD1. The optimal permeabilization time should be determined empirically.

PRKD1 antibodies have been successfully validated for immunofluorescence applications in HeLa and LNCaP cell lines, with recommended dilutions ranging from 1:50 to 1:500 . For optimal results, researchers should follow manufacturer-specific protocols, such as those provided by Proteintech for their PRKD1 antibody (83174-5-RR) .

What are the key differences between polyclonal and monoclonal PRKD1 antibodies for research applications?

The choice between polyclonal and monoclonal PRKD1 antibodies depends on the specific research application and experimental requirements:

How does HRP conjugation affect the performance and application range of PRKD1 antibodies?

HRP (horseradish peroxidase) conjugation directly links the enzyme to PRKD1 antibodies, offering several advantages and limitations:

Advantages of HRP-conjugated PRKD1 antibodies:

• Simplified Protocols: Eliminates the need for secondary antibody incubation steps, reducing experimental time and potential background

• Enhanced Sensitivity: Direct conjugation can improve detection sensitivity in certain applications

• Reduced Cross-Reactivity: Minimizes potential cross-reactivity issues associated with secondary antibodies

• Multiplexing Capability: Facilitates simultaneous detection of multiple targets in co-staining experiments

Limitations and Considerations:

• Signal Amplification: HRP-conjugated antibodies lack the signal amplification provided by secondary antibody systems (where multiple secondary antibodies can bind each primary)

• Application Restrictions: HRP-conjugated PRKD1 antibodies are primarily optimized for ELISA applications , though they can be used in other applications with optimization

• Storage Sensitivity: HRP conjugates may have reduced shelf-life compared to unconjugated antibodies and require protection from light

• Buffer Compatibility: HRP activity can be affected by certain buffer components, requiring careful consideration of experimental conditions

The Qtonics HRP-conjugated PRKD1 antibody (QA55732) is supplied in a preservative buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . This formulation helps maintain both antibody integrity and HRP enzymatic activity during storage.

How can researchers investigate the molecular mechanisms of PRKD1-mediated regulation of the β-catenin/TCF-4 transcription complex?

Investigating PRKD1's regulation of the β-catenin/TCF-4 transcription complex requires multifaceted experimental approaches. Based on research showing that PRKD1 attenuates tumorigenesis in colon cancer by modulating this complex , the following methodological strategies are recommended:

• Co-Immunoprecipitation Studies: Extract equal amounts of protein from nuclear lysates of cells with differential PRKD1 expression (e.g., SW480-PKD1-GFP vs. SW480-GFP controls). Immunoprecipitate using anti-TCF-4 antibody, then probe for β-catenin, PKD1, and TCF-4 by Western blotting to assess complex formation .

• Chromatin Immunoprecipitation (ChIP): Perform ChIP assays to investigate how PRKD1 affects β-catenin/TCF-4 binding to target gene promoters. This approach can reveal the direct impact of PRKD1 on transcriptional regulation.

• Luciferase Reporter Assays: Employ TCF/LEF reporter constructs to quantitatively assess how PRKD1 manipulation affects β-catenin-dependent transcriptional activity.

• Protein Phosphorylation Analysis: Investigate whether PRKD1 directly phosphorylates β-catenin or TCF-4 using in vitro kinase assays and phospho-specific antibodies. Identify phosphorylation sites using mass spectrometry.

• Proximity Ligation Assay (PLA): Use PLA to visualize and quantify protein-protein interactions between PRKD1, β-catenin, and TCF-4 in situ, providing spatial context to these molecular interactions.

Research has demonstrated that PRKD1 overexpression leads to decreased formation of the β-catenin/TCF-4 transcription complex in the nucleus, thereby attenuating oncogenic Wnt signaling in colon cancer cells .

What approaches can be used to study the epigenetic regulation of PRKD1 expression in different cancer types?

PRKD1 expression is frequently silenced through epigenetic mechanisms in cancer, making the study of its epigenetic regulation particularly relevant. Based on research demonstrating aberrant methylation of the PRKD1 promoter in invasive cancer , several methodological approaches are recommended:

• Methylation Analysis Techniques:

  • Reduced representation bisulfite deep sequencing for genome-wide methylation profiling

  • Methylation-specific PCR (MSP-PCR) for targeted analysis of PRKD1 promoter methylation

  • In situ MSP-PCR for cellular visualization of methylation patterns

• DNA Methyltransferase Inhibitor Studies: Treat cancer cell lines with demethylating agents like decitabine to assess PRKD1 reexpression. This approach has successfully demonstrated restoration of PKD1 expression and inhibition of tumor spread in both in vitro and in vivo models .

• Chromatin Immunoprecipitation (ChIP): Use ChIP to analyze histone modifications associated with the PRKD1 promoter, including repressive (H3K27me3, H3K9me3) and activating (H3K4me3, H3K27ac) marks.

• Expression Analysis Following Epigenetic Modulation:

  • RT-PCR to detect PRKD1 mRNA expression changes

  • Western blotting to assess protein level changes

  • Immunohistochemistry to visualize expression in tissue samples

• Clinical Correlation Studies: Analyze PRKD1 promoter methylation across cancer types and stages to establish patterns. Previous research has examined methylation in various breast cancer subtypes, from normal tissue to invasive carcinomas .

PCR reactions for analyzing PRKD1 expression can be conducted under conditions of 1 minute at 55°C and a 1-minute extension at 72°C for 35 cycles .

How can researchers design experiments to investigate the role of PRKD1 in the polyubiquitin-dependent proteasomal degradation of MTA1?

PRKD1 has been shown to regulate MTA1 through polyubiquitin-dependent proteasomal degradation, representing a key mechanism for its metastasis-suppressive function . To investigate this process, researchers can employ the following methodological approaches:

• Ubiquitination Assays: Perform in vivo ubiquitination assays by co-expressing PRKD1, MTA1, and HA-tagged ubiquitin in cells, followed by immunoprecipitation of MTA1 and Western blotting for HA to detect ubiquitinated species.

• Proteasome Inhibition Studies: Treat cells expressing PRKD1 and MTA1 with proteasome inhibitors (e.g., MG132) to determine if PRKD1-mediated reduction in MTA1 levels is reversed, confirming proteasome-dependent degradation.

• Domain Mapping Experiments: Generate PRKD1 domain mutants to identify which regions are essential for MTA1 binding and subsequent degradation. Research has shown that PRKD1 utilizes its N-terminal and kinase domains to effectively inhibit MTA1 levels .

• Phosphorylation Site Identification: Use site-directed mutagenesis to identify MTA1 residues phosphorylated by PRKD1 that trigger ubiquitination and degradation. Confirm using phospho-specific antibodies or mass spectrometry.

• Live-Cell Imaging: Employ fluorescently tagged MTA1 to monitor its real-time degradation in response to PRKD1 expression or activation.

• E3 Ligase Identification: Use proteomics approaches to identify the E3 ubiquitin ligase responsible for MTA1 ubiquitination following PRKD1-mediated phosphorylation.

Research has established that PRKD1-mediated downregulation of MTA1 results in significant suppression of prostate cancer progression and metastasis in physiologically relevant spontaneous tumor models .

How can researchers validate the specificity of PRKD1 antibodies for their particular experimental system?

Ensuring antibody specificity is crucial for generating reliable PRKD1 research data. Comprehensive validation approaches include:

• Genetic Controls:

  • PRKD1 knockout cell lines (CRISPR/Cas9-generated)

  • siRNA or shRNA knockdown of PRKD1

  • PRKD1 overexpression systems (e.g., SW480-PKD1-GFP)

• Peptide Competition Assays: Pre-incubate the PRKD1 antibody with the immunizing peptide before application to samples. The Qtonics antibody, for example, uses a recombinant human serine/threonine-protein kinase D1 protein fragment (residues 674-904AA) as immunogen . Signal abolishment confirms specificity.

• Multiple Antibody Validation: Compare results using antibodies raised against different PRKD1 epitopes. The search results mention several commercially available options with different characteristics:

  • Proteintech antibody (83174-5-RR): Rabbit IgG, recombinant class

  • Qtonics antibody (QA55732): Rabbit polyclonal, HRP-conjugated

  • Antibodies.com product (A38003): Rabbit polyclonal

• Western Blot Analysis: Verify single band detection at the expected molecular weight (102 kDa) , with additional bands potentially indicating non-specific binding or PRKD1 isoforms.

• Cross-Reactivity Assessment: Test antibodies against related family members (PRKD2, PRKD3) to ensure specificity for PRKD1.

• Lot-to-Lot Consistency Testing: When receiving new antibody lots, perform side-by-side comparisons with previous lots to ensure consistent performance.

These validation steps should be performed for each specific application (WB, IF, IHC, FC) as antibody performance can vary between applications.

What are the common sources of false positives and false negatives when using PRKD1 antibodies, and how can they be mitigated?

Understanding potential sources of false results is essential for accurate PRKD1 research:

Sources of False Positives:

• Cross-reactivity with related kinases: PRKD1 shares sequence homology with PRKD2 and PRKD3. Mitigation: Use antibodies validated for specificity against related family members.

• Non-specific binding: Secondary antibodies may bind non-specifically to endogenous immunoglobulins. Mitigation: Include isotype controls and consider using directly conjugated antibodies like HRP-conjugated PRKD1 antibodies .

• Autofluorescence in IF/IHC: Certain fixatives or tissues produce autofluorescence. Mitigation: Include unstained controls and consider autofluorescence quenching steps.

• Protein overexpression artifacts: Overexpressed PRKD1 may localize differently than endogenous protein. Mitigation: Validate findings using multiple approaches and examine endogenous protein.

Sources of False Negatives:

• Epitope masking: Protein-protein interactions or post-translational modifications may mask antibody binding sites. Mitigation: Try multiple antibodies targeting different PRKD1 epitopes.

• Protein degradation: Improper sample handling can lead to PRKD1 degradation. Mitigation: Use fresh samples and appropriate protease inhibitors.

• Suboptimal fixation: In IF/IHC, inappropriate fixation can destroy epitopes. Mitigation: Optimize fixation protocols and consider antigen retrieval methods.

• Low PRKD1 expression: Epigenetic silencing may reduce PRKD1 below detection limits in some cancer cells . Mitigation: Consider demethylating agent treatment to restore expression.

• Incorrect antibody dilution: Too dilute antibody solutions may yield false negatives. Mitigation: Titrate antibodies to determine optimal concentration for each application and cell type .

How can researchers resolve contradictory results between PRKD1 expression analysis at the mRNA versus protein levels?

Discrepancies between mRNA and protein expression of PRKD1 are not uncommon and can yield important biological insights. Systematic approaches to resolve such contradictions include:

• Comprehensive Technical Validation:

  • Confirm primer specificity for PRKD1 mRNA detection

  • Validate antibody specificity for PRKD1 protein detection using methods outlined in Q7.1

  • Include appropriate positive and negative controls in both assays

• Post-transcriptional Regulation Assessment:

  • Examine PRKD1 mRNA stability through actinomycin D chase experiments

  • Investigate miRNA-mediated regulation of PRKD1 using miRNA prediction tools and validation experiments

  • Analyze alternative splicing of PRKD1 through exon-specific PCR

• Post-translational Regulation Analysis:

  • Assess protein stability through cycloheximide chase experiments

  • Investigate ubiquitin-mediated proteasomal degradation using proteasome inhibitors

  • Examine PRKD1 protein half-life in different cellular contexts

• Epigenetic Regulation:

  • Analyze PRKD1 promoter methylation status, as it is frequently methylated in cancer cells

  • Consider treatment with demethylating agents to restore expression

• Translational Efficiency:

  • Employ polysome profiling to assess translational efficiency of PRKD1 mRNA

  • Examine 5' and 3' UTR regulatory elements that might affect translation

Research has shown that PRKD1 expression can be silenced at the epigenetic level in invasive cancers , and the protein can be regulated post-translationally through mechanisms like polyubiquitin-dependent proteasomal degradation . Understanding these regulatory layers is essential for interpreting contradictory results between mRNA and protein analyses.

What are promising approaches for developing highly specific inhibitors or activators of PRKD1 for research applications?

The development of specific PRKD1 modulators represents an important frontier in both basic research and potential therapeutic applications. Promising approaches include:

• Structure-Based Drug Design: Utilizing the crystal structure of PRKD1's catalytic domain to design small molecules that selectively bind PRKD1 over related kinases. This approach requires detailed structural information about the ATP-binding pocket and regulatory domains.

• Allosteric Modulators: Targeting unique allosteric sites on PRKD1 rather than the highly conserved ATP-binding pocket can potentially provide greater selectivity. These modulators can be identified through high-throughput screening or computational modeling.

• Peptide-Based Inhibitors: Developing peptides that mimic PRKD1 interaction surfaces with key binding partners like MTA1 . These peptides can be designed to specifically disrupt protein-protein interactions critical for PRKD1 function.

• Antibody-Based Approaches: Engineering function-modulating antibodies or antibody fragments that specifically bind to PRKD1. While conventional antibodies like those described in the search results are used primarily for detection , engineered variants could potentially modulate PRKD1 activity.

• Epigenetic Modulators: Given PRKD1's epigenetic silencing in invasive cancers , compounds that selectively reverse PRKD1 promoter methylation could serve as indirect activators of PRKD1 expression and function.

• PROTAC Technology: Proteolysis-targeting chimeras (PROTACs) that selectively target PRKD1 for degradation could provide another approach for functional modulation in research settings.

How might single-cell analysis technologies advance our understanding of PRKD1's role in heterogeneous tumor populations?

Single-cell technologies offer unprecedented insights into cellular heterogeneity and can revolutionize our understanding of PRKD1's role in cancer:

• Single-Cell RNA Sequencing (scRNA-seq):

  • Reveals heterogeneity in PRKD1 expression across individual cells within tumors

  • Identifies co-expression patterns between PRKD1 and other genes/pathways

  • Characterizes transcriptional states associated with PRKD1 expression or silencing

  • Enables trajectory analysis to map PRKD1 expression changes during cancer progression

• Single-Cell Proteomics:

  • Mass cytometry (CyTOF) with PRKD1 antibodies allows simultaneous detection of PRKD1 and dozens of other proteins at the single-cell level

  • Reveals correlations between PRKD1 protein levels and activation states of other signaling pathways

• Single-Cell Epigenomics:

  • Single-cell ATAC-seq or single-cell methylation analysis can reveal the relationship between chromatin accessibility, DNA methylation, and PRKD1 expression in individual cells

  • Particularly relevant given PRKD1's epigenetic silencing in invasive cancers

• Spatial Transcriptomics/Proteomics:

  • Maps PRKD1 expression within the spatial context of the tumor microenvironment

  • Correlates PRKD1 levels with cell-cell interactions and microenvironmental factors

• Live-Cell Imaging at Single-Cell Resolution:

  • Fluorescent reporter systems to track PRKD1 expression, localization, and activity in real-time

  • Reveals dynamic changes in PRKD1 behavior during cell division, migration, and response to therapeutic agents

These approaches could help resolve contradictory findings in bulk tumor analyses and identify specific cell populations where PRKD1 plays critical roles in tumor suppression or progression.

What are the emerging connections between PRKD1 and immune regulation that warrant investigation in cancer research?

While the direct search results don't explicitly address PRKD1's role in immune regulation, this represents an emerging area worthy of investigation based on broader knowledge of kinase signaling in immune responses. Potential research directions include:

• PRKD1 Expression in Immune Cell Populations:

  • Characterize PRKD1 expression patterns across different immune cell types (T cells, B cells, macrophages, dendritic cells)

  • Investigate how PRKD1 expression in immune cells changes in the tumor microenvironment

• PRKD1's Role in Immune Cell Function:

  • Examine how PRKD1 modulation affects T cell activation, cytokine production, and anti-tumor responses

  • Investigate PRKD1's influence on macrophage polarization (M1 vs. M2) within the tumor microenvironment

  • Study how PRKD1 expression in cancer cells affects their interaction with immune cells

• Integration with Immune Checkpoint Pathways:

  • Explore potential connections between PRKD1 signaling and immune checkpoint molecules (PD-1/PD-L1, CTLA-4)

  • Investigate whether PRKD1 restoration in cancer cells affects their susceptibility to immune checkpoint blockade

• Epigenetic Regulation in Immune Contexts:

  • Given PRKD1's epigenetic silencing in cancer , explore whether similar mechanisms operate in immune cells within the tumor microenvironment

  • Investigate how demethylating agents affect both PRKD1 expression and immune cell function simultaneously

• Cytokine Signaling Crosstalk:

  • Characterize potential crosstalk between PRKD1 signaling pathways and cytokine signaling networks relevant to anti-tumor immunity

  • Explore whether PRKD1-mediated regulation of β-catenin or MTA1 intersects with immune regulatory pathways

These investigations could potentially reveal new dimensions of PRKD1's tumor-suppressive functions through immune-mediated mechanisms, opening avenues for combinatorial therapeutic approaches targeting both cancer cells and the immune microenvironment.

How can researchers integrate multi-omics data to better understand PRKD1's role in cancer progression?

Multi-omics integration provides a comprehensive view of PRKD1's regulatory networks and functional impact in cancer. Methodological approaches include:

• Integrated Genomic and Epigenomic Analysis:

  • Correlate PRKD1 promoter methylation with gene expression across cancer types

  • Identify genetic alterations (mutations, copy number variations) affecting PRKD1 and correlate with expression

  • Investigate chromatin accessibility at the PRKD1 locus using ATAC-seq data

• Transcriptomic Integration:

  • Perform differential gene expression analysis between PRKD1-high and PRKD1-low tumors

  • Conduct gene set enrichment analysis (GSEA) to identify pathways correlated with PRKD1 expression

  • Quantify PRKD1 mRNA using exon-level analysis as described in previous research: "PCR primers were designed using the template regions recommended by SnowShoes-FTD. The gene expression levels were calculated as the sum of the individual exon read counts and exon junction read counts"

• Proteomic Correlation:

  • Correlate PRKD1 protein levels with global proteome changes

  • Identify phosphorylation networks affected by PRKD1 expression

  • Study how PRKD1-mediated post-translational modifications alter protein stability (e.g., MTA1 degradation)

• Metabolomic Integration:

  • Investigate metabolic pathways affected by PRKD1 expression

  • Correlate metabolomic signatures with PRKD1 status in tumors

• Clinical Data Correlation:

  • Integrate PRKD1 multi-omics data with patient survival, treatment response, and metastatic potential

  • Research has shown that "progression of human prostate tumours to increased invasiveness was accompanied by decreased and increased levels of PKD1 and MTA1, respectively"

• Network Analysis:

  • Construct protein-protein interaction networks centered on PRKD1

  • Include known interactions such as the PRKD1-MTA1 regulatory axis and the β-catenin/TCF-4 transcription complex

These integrative approaches can reveal context-dependent functions of PRKD1 and identify potential biomarkers or therapeutic targets for personalized medicine.

What bioinformatic approaches can be used to predict novel PRKD1 substrates and interacting partners?

Identifying novel PRKD1 substrates and interacting partners can significantly advance our understanding of its biological functions. Computational approaches include:

• Consensus Phosphorylation Motif Analysis:

  • Utilize known PRKD1 substrates to define consensus phosphorylation motifs

  • Scan proteome databases for proteins containing these motifs

  • Prioritize candidates based on cellular compartmentalization and biological context

• Protein-Protein Interaction Prediction:

  • Employ structure-based docking simulations to predict potential PRKD1 binding partners

  • Utilize machine learning algorithms trained on known kinase-substrate interactions

  • Consider domain-specific interactions, focusing on PRKD1's N-terminal and kinase domains which are known to be important for MTA1 regulation

• Co-expression Network Analysis:

  • Analyze large-scale gene expression datasets to identify genes consistently co-expressed with PRKD1

  • Use weighted gene correlation network analysis (WGCNA) to identify modules associated with PRKD1 expression

• Evolutionary Conservation Analysis:

  • Identify proteins with conserved potential PRKD1 phosphorylation sites across species

  • Higher conservation suggests functional importance and increases confidence in prediction

• Text Mining and Literature-Based Discovery:

  • Employ natural language processing to identify proteins mentioned in conjunction with PRKD1 in scientific literature

  • Discover implicit connections through shared intermediate interactors

• Integration of Phosphoproteomic Data:

  • Analyze phosphoproteomic datasets from cells with manipulated PRKD1 expression or activity

  • Identify phosphorylation sites that change in response to PRKD1 modulation

These computational predictions should subsequently be validated experimentally using approaches like in vitro kinase assays, co-immunoprecipitation, and proximity ligation assays, as demonstrated in previous PRKD1 research .

How can researchers design comprehensive experiments to elucidate the reciprocal regulation between PRKD1 and β-catenin signaling in cancer?

The reciprocal regulation between PRKD1 and β-catenin signaling represents an important area for cancer research, particularly given evidence that PRKD1 attenuates tumorigenesis by modulating the β-catenin/TCF-4 transcription complex . A comprehensive experimental design should include:

• Bidirectional Expression Modulation:

  • Generate cell line panels with: PRKD1 overexpression, PRKD1 knockdown, β-catenin overexpression, β-catenin knockdown, and combinations thereof

  • Compare SW480-PKD1-GFP cells with SW480-GFP control cells as previously described

  • Utilize inducible expression systems to study temporal aspects of this regulation

• Transcriptional Activity Analysis:

  • Employ TOPFlash/FOPFlash reporter assays to quantify β-catenin/TCF transcriptional activity

  • Perform RNA-seq to identify global transcriptional changes mediated by the PRKD1-β-catenin axis

  • Use ChIP-seq to map genome-wide binding sites of β-catenin/TCF-4 in the presence or absence of PRKD1

• Protein-Protein Interaction Studies:

  • Perform co-immunoprecipitation experiments as previously described: "equal amounts of protein extracted from the nuclear lysates of SW480-PKD1-GFP or SW480-GFP cells were subjected to immuno-precipitation using anti-TCF-4 antibody"

  • Employ proximity ligation assays to visualize these interactions in situ

  • Use FRET/BRET assays to study dynamic interactions in living cells

• Subcellular Localization Analysis:

  • Fractionate cells into nuclear and cytoplasmic compartments to quantify β-catenin distribution

  • Perform immunofluorescence microscopy to visualize localization changes

  • Use live-cell imaging to track real-time changes in protein localization

• Phosphorylation Site Mapping:

  • Identify PRKD1-dependent phosphorylation sites on β-catenin using mass spectrometry

  • Generate phospho-mimetic and phospho-deficient mutants to assess functional significance

  • Develop phospho-specific antibodies for identified sites

• In Vivo Validation:

  • Utilize xenograft models expressing different combinations of PRKD1 and β-catenin

  • Analyze patient-derived organoids to validate findings in more physiologically relevant systems

  • Correlate PRKD1 and β-catenin expression patterns in human tumor samples

Previous research has established that "a lower level of TCF-4 and β-catenin and therefore lower β-catenin/TCF-4 transcription complex was detected in the PKD1 overexpressing cells compared to the control cells" , providing a foundation for these more comprehensive investigations.

What minimum validation standards should researchers apply when publishing studies using PRKD1 antibodies?

To ensure reproducibility and reliability of PRKD1 research, the following minimum validation standards should be implemented:

• Antibody Validation:

  • Report complete antibody information: manufacturer, catalog number, lot number, RRID (Research Resource Identifier, e.g., AB_3670868 ), host species, clonality, and immunogen

  • Demonstrate antibody specificity using at least two independent methods (e.g., Western blot plus genetic knockdown/knockout)

  • Include positive controls from cells known to express PRKD1 (e.g., HeLa or LNCaP cells )

  • Document optimization steps for each application, including dilution optimization

• Experimental Design:

  • Include biological replicates (minimum n=3) and technical replicates

  • Implement appropriate statistical analysis with clearly stated tests and significance criteria

  • Include both positive and negative controls for each experiment

  • Report all relevant experimental conditions (e.g., cell culture conditions, treatment durations, antibody incubation times and temperatures)

• Results Reporting:

  • Present full, uncropped Western blot images with molecular weight markers visible

  • For immunofluorescence, include appropriate controls and scale bars

  • Show representative images alongside quantification from multiple experiments

  • Disclose any image processing or enhancement steps

• Protocol Transparency:

  • Provide detailed, step-by-step protocols or reference standardized protocols (e.g., "FC protocol for PRKD1 antibody 83174-5-RR" )

  • Report buffer compositions, incubation times, and washing steps

  • Disclose any deviations from manufacturer recommendations

• Data Availability:

  • Deposit raw data in appropriate repositories

  • Make detailed protocols available through protocol sharing platforms

  • Consider using electronic lab notebooks to enhance reproducibility

These standards align with broader initiatives to enhance reproducibility in antibody-based research and ensure that findings regarding PRKD1's functions in cancer can be reliably built upon by the research community.

How should researchers approach conflicting results about PRKD1's role in different cancer contexts?

PRKD1 may exhibit context-dependent functions across different cancer types or even within different stages of the same cancer. Resolving conflicting results requires systematic approaches:

• Contextual Analysis:

  • Carefully document the specific cancer type, subtype, and stage being studied

  • Consider the genetic background of cell lines or patient samples (mutation status of key oncogenes/tumor suppressors)

  • Evaluate the microenvironmental context (2D vs. 3D culture, presence of stromal components)

• Expression Level Considerations:

  • Quantify baseline PRKD1 expression levels in each experimental system

  • Consider that PRKD1 is epigenetically silenced in some invasive cancers , potentially explaining different functional outcomes

  • Distinguish between studies of PRKD1 loss vs. PRKD1 restoration/overexpression

• Pathway Integration:

  • Assess the status of interacting pathways that might modulate PRKD1 function:

    • β-catenin/TCF-4 signaling status

    • MTA1 expression levels

    • Other relevant signaling pathways not directly addressed in the search results

• Technical Reconciliation:

  • Compare methodologies used (antibodies, detection methods, expression modulation approaches)

  • Evaluate whether differences in experimental techniques could explain discrepancies

  • Consider performing side-by-side experiments using multiple methodologies

• Integrative Validation:

  • Validate findings across multiple model systems (cell lines, organoids, animal models)

  • Correlate in vitro findings with patient data

  • Consider collaborative studies to independently validate findings

Research has shown that PRKD1 can function as a tumor suppressor by attenuating β-catenin/TCF-4 signaling in colon cancer and by promoting degradation of metastasis-associated protein 1 in prostate cancer . Understanding the molecular context of these regulatory mechanisms may help reconcile seemingly conflicting results across cancer types.

What considerations should guide the development of standardized protocols for PRKD1 antibody-based assays in multi-center studies?

Standardization of PRKD1 antibody-based assays for multi-center studies requires careful attention to several key considerations:

• Antibody Selection and Validation:

  • Choose antibodies with robust validation data across multiple applications

  • Consider recombinant antibodies when available, as they offer reduced lot-to-lot variability compared to conventional polyclonal antibodies

  • Select antibodies with clear documentation of specificity, such as the Proteintech PRKD1 antibody (83174-5-RR) which has been validated in multiple cell lines

• Protocol Standardization:

  • Develop detailed, step-by-step protocols for each application (WB, IF, IHC, FC)

  • Specify critical parameters including:

    • Sample preparation methods

    • Antibody dilutions (e.g., 1:50-1:500 for IF/ICC applications )

    • Incubation times and temperatures

    • Buffer compositions

    • Detection methods

• Quality Control Measures:

  • Implement centralized antibody procurement and validation

  • Distribute reference standards (e.g., cell lysates with known PRKD1 expression levels)

  • Include inter-laboratory controls to assess consistency

  • Establish acceptance criteria for assay performance

• Training and Proficiency Testing:

  • Develop training materials for all participating laboratories

  • Conduct initial proficiency testing to ensure comparable results across sites

  • Implement periodic reassessment to maintain consistency

• Data Collection and Analysis Standardization:

  • Establish uniform data collection templates

  • Standardize image acquisition parameters

  • Implement centralized or standardized data analysis methods

  • Define quantification metrics and cutoff values

• Documentation Requirements:

  • Maintain detailed records of protocol deviations

  • Document reagent lot numbers and expiration dates

  • Record environmental conditions that may affect results

  • Implement a centralized database for tracking all experimental variables