Phospho-PRKCD (Tyr313) Antibody

What is PRKCD and why is the Tyr313 phosphorylation site significant?

PRKCD (Protein Kinase C delta) belongs to the PKC family of serine/threonine kinases that are activated intracellularly through signal transduction pathways. In humans, at least 12 different PKC polypeptides have been identified, each differing in structure, distribution, localization, and substrate specificity. The Tyr313 site is located in the hinge region of PRKCD, making it a structurally significant phosphorylation site. This site has been implicated in cancer progression, particularly in triple-negative breast cancer (TNBC), where PRKCD_pY313 has been shown to inhibit apoptosis and promote migration and invasion of cancer cells . Research indicates that high PRKCD expression correlates with poor survival, making it a valuable target for cancer research .

What are the key specifications of commercially available Phospho-PRKCD (Tyr313) antibodies?

Commercial Phospho-PRKCD (Tyr313) antibodies are typically rabbit polyclonal antibodies that detect endogenous levels of PKC delta only when phosphorylated at Tyrosine 313 . These antibodies are generally available in unconjugated form and demonstrate reactivity with human, mouse, and rat samples . The antibodies are commonly supplied at a concentration of 1 mg/mL in formulations containing PBS, 50% glycerol, 0.5% BSA, and 0.02% sodium azide for stability . They are suitable for multiple applications including Western Blot (recommended dilution 1:500-1:2000), Immunohistochemistry (1:100-1:300), Immunofluorescence (1:200-1:1000), and ELISA (1:10000) .

How specific is the Phospho-PRKCD (Tyr313) antibody?

The Phospho-PRKCD (Tyr313) antibody demonstrates high specificity, detecting endogenous levels of PKC delta only when phosphorylated at Tyrosine 313 . The antibody is typically produced against a synthesized peptide derived from human PKC delta around the phosphorylation site of Tyr313, specifically within the amino acid range 279-328 . This site-specific design ensures that the antibody can distinguish between phosphorylated and non-phosphorylated forms of the protein, making it valuable for studying activation states of PRKCD in various biological contexts. The specificity of these antibodies is confirmed through validation studies using phosphorylated and non-phosphorylated protein controls.

What experimental applications are most suitable for Phospho-PRKCD (Tyr313) antibody?

The Phospho-PRKCD (Tyr313) antibody can be effectively utilized in multiple experimental applications. Western blotting represents a primary application, allowing for detection and quantification of phosphorylated PRKCD at Tyr313 in cell and tissue lysates. Immunohistochemistry applications enable visualization of phosphorylated PRKCD in tissue sections, providing insights into spatial distribution and expression levels in physiological and pathological states. Immunofluorescence techniques offer higher resolution imaging of subcellular localization. ELISA applications provide quantitative measurement of phosphorylated PRKCD levels . For cancer research specifically, this antibody has proven valuable in studying the role of PRKCD_pY313 in triple-negative breast cancer progression, where phosphorylation at this site correlates with tumor invasiveness and metastatic potential .

How should researchers design experiments to study PRKCD_pY313 in cancer progression?

When designing experiments to investigate PRKCD_pY313 in cancer progression, researchers should consider a multi-faceted approach. Begin with expression analysis comparing PRKCD_pY313 levels between cancer and normal tissues using Western blot or immunohistochemistry. For functional studies, employ gain-of-function and loss-of-function approaches by overexpressing wild-type PRKCD, phosphomimetic mutants, or phospho-deficient mutants (such as Y313F) . Assess cellular effects through proliferation assays, invasion assays, apoptosis assays, and mitochondrial function tests. Include measurement of reactive oxygen species (ROS) levels, as PRKCD_pY313 has been shown to reduce ROS levels in TNBC cell lines . For signaling pathway analysis, evaluate downstream effects on other kinases, particularly Src_pY419 and p38_pT180/pY182, which have been shown to be upregulated by PRKCD_pY313 . Finally, validate findings in vivo using xenograft models to assess tumor progression, accompanied by immunohistochemical analysis of markers like Ki-67, Bcl-xl, Vimentin, Bad, cleaved caspase 3, and ZO1.

What controls should be included when using Phospho-PRKCD (Tyr313) antibody?

When using Phospho-PRKCD (Tyr313) antibody, several critical controls should be included to ensure experimental validity. Positive controls should include samples known to express high levels of phosphorylated PRKCD at Tyr313, such as stimulated cancer cell lines. Negative controls should include samples where PRKCD phosphorylation is inhibited, such as cells treated with appropriate kinase inhibitors or phosphatase-treated lysates. Specificity controls should include the use of blocking peptides corresponding to the phosphorylated epitope, which should abolish antibody binding. For genetic validation, researchers should include PRKCD knockdown samples or cells expressing the phospho-deficient Y313F mutant as negative controls . For loading controls in Western blots, housekeeping proteins such as beta-actin or GAPDH should be used, similar to those included in commercial phospho-antibody arrays . Additionally, when performing immunohistochemistry or immunofluorescence, include secondary antibody-only controls to assess non-specific binding.

How does PRKCD_pY313 influence cancer cell signaling networks?

PRKCD_pY313 plays a complex role in cancer cell signaling networks, particularly in TNBC. Phosphorylation at Y313 significantly upregulates Src_pY419 and p38_pT180/pY182, suggesting its involvement in activating these critical oncogenic pathways . This creates a potential signaling cascade where PRKCD_pY313 serves as an upstream regulator of Src activation, which in turn mediates cancer cell proliferation, invasion, and metastasis. Additionally, PRKCD_pY313 impacts mitochondrial membrane potential and reduces reactive oxygen species (ROS) levels, suggesting a role in metabolic reprogramming and oxidative stress responses in cancer cells . The effects on apoptotic machinery are evidenced by altered expression of Bcl-xl (increased) and Bad (decreased), indicating that PRKCD_pY313 promotes cell survival by modulating the balance between pro-survival and pro-apoptotic factors . Furthermore, the impact on ZO1 and Vimentin expression suggests involvement in epithelial-to-mesenchymal transition, a critical process in cancer metastasis.

How can researchers distinguish between different phosphorylation sites on PRKCD?

Distinguishing between different phosphorylation sites on PRKCD requires a combination of techniques and careful experimental design. Site-specific phospho-antibodies, such as those against PRKCD_pY313, provide the most direct approach by selectively recognizing specific phosphorylated residues . For comprehensive phosphorylation profiling, researchers should consider phosphoproteomics approaches using mass spectrometry following enrichment with techniques like Superbinder resin for tyrosine phosphorylation and TiO2 columns for serine/threonine phosphorylation . Mutational analysis using site-directed mutagenesis to generate phospho-deficient mutants (e.g., Y313F) or phosphomimetic mutants can validate findings and assess the functional significance of specific phosphorylation sites . Researchers should also consider temporal dynamics of phosphorylation events using time-course experiments with various stimuli. Computational approaches including bioinformatics analysis of phosphorylation motifs and structural modeling can predict the impact of phosphorylation on protein conformation and interactions.

What is the relationship between PRKCD_pY313 and Src kinase in cancer progression?

The relationship between PRKCD_pY313 and Src kinase represents a critical signaling axis in cancer progression, particularly in TNBC. Research has demonstrated that PRKCD_pY313 significantly upregulates Src_pY419, a key activation marker of Src kinase . This suggests a direct or indirect regulatory relationship where phosphorylation of PRKCD at Y313 leads to Src activation. Functionally, this signaling axis promotes cancer cell proliferation, invasion, and metastasis. The clinical significance of this relationship is underscored by the finding that Dasatinib, a Src family kinase inhibitor, significantly inhibits the growth of cells overexpressing PRKCD_pY313 . This therapeutic vulnerability suggests that targeting the PRKCD_pY313-Src axis could be a viable strategy for treating TNBC. Further evidence comes from xenograft models where PRKCD_pY313 promoted tumor progression with increased markers of proliferation (Ki-67) and anti-apoptotic factors (Bcl-xl), while the phospho-deficient Y313F mutant showed the opposite effects . The combined inhibition of both Src (with Dasatinib) and p38 (with Adezmapimod) showed enhanced therapeutic effects, highlighting the interconnected nature of these signaling pathways downstream of PRKCD_pY313.

What are the optimal storage and handling conditions for Phospho-PRKCD (Tyr313) antibody?

For optimal performance, Phospho-PRKCD (Tyr313) antibodies should be stored at -20°C for up to one year from the date of receipt . Avoid repeated freeze-thaw cycles as they can compromise antibody activity and specificity. When handling the antibody, always work with clean pipettes and tubes to prevent contamination. Antibodies are typically formulated in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide for stability . For working solutions, dilute only the amount needed for immediate use in appropriate buffers free of contaminants. After dilution, store working solutions at 4°C for short-term use (1-2 weeks). For long-term storage of diluted antibodies, add carrier proteins such as BSA (0.1-1%) to prevent adhesion to tube walls and maintain stability. Always centrifuge the antibody vial before opening to ensure recovery of all liquid, especially after shipment or storage at 4°C.

What are common troubleshooting strategies for Western blot applications using this antibody?

When troubleshooting Western blot applications with Phospho-PRKCD (Tyr313) antibody, several strategies can help optimize results. For weak or no signal, verify phosphorylation status by ensuring samples were properly stimulated or treated to induce PRKCD phosphorylation at Tyr313. Consider including positive controls such as lysates from cells known to express high levels of PRKCD_pY313. Optimize antibody concentration by testing a range of dilutions (1:500-1:2000 is recommended) . For high background, increase blocking time or concentration, optimize secondary antibody dilution, and ensure thorough washing between steps. For non-specific bands, increase the stringency of wash buffers by adjusting salt concentration or adding mild detergents. When detecting endogenous PRKCD_pY313, sample preparation is critical - use phosphatase inhibitors in lysis buffers to preserve phosphorylation status. Consider enrichment techniques like immunoprecipitation for low-abundance phosphoproteins before Western blotting. For quantitative analysis, ensure linear range detection and use appropriate loading controls like beta-actin or GAPDH .

How can researchers optimize immunohistochemistry protocols for Phospho-PRKCD (Tyr313) antibody?

Optimizing immunohistochemistry protocols for Phospho-PRKCD (Tyr313) antibody requires careful attention to several key factors. Begin with appropriate antigen retrieval methods - heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is typically effective for phospho-epitopes. Test different retrieval times and temperatures to determine optimal conditions. For antibody concentration, start with the recommended dilution range (1:100-1:300) and perform a dilution series to identify the optimal concentration that maximizes specific staining while minimizing background. Blocking is critical - use 5-10% normal serum from the same species as the secondary antibody for 1-2 hours at room temperature. Include phosphatase inhibitors in all buffers to preserve phosphorylation status. For detection systems, consider signal amplification methods like tyramide signal amplification for low-abundance phospho-proteins. Controls should include phosphatase-treated sections to confirm phospho-specificity and tissues with known PRKCD_pY313 expression as positive controls. For dual staining to co-localize with other markers, use sequential rather than simultaneous immunostaining to minimize cross-reactivity. Finally, for quantitative analysis, use digital image analysis software to measure staining intensity objectively.

How does inhibition of PRKCD_pY313 affect cancer cell behavior in experimental models?

Inhibition of PRKCD_pY313 demonstrates significant anti-cancer effects across multiple experimental models. In cellular models, reduced PRKCD_pY313 levels or expression of the phospho-deficient Y313F mutant reverses the oncogenic properties observed with wild-type PRKCD, including decreased proliferation, invasion, and metastatic potential . At the molecular level, inhibition of PRKCD_pY313 significantly downregulates Src_pY419 and p38_pT180/pY182, disrupting these critical oncogenic signaling pathways . Metabolically, low PRKCD_pY313 decreases mitochondrial membrane potential and increases reactive oxygen species (ROS) levels, potentially enhancing cancer cell susceptibility to oxidative stress-induced death . In pharmacological approaches, Dasatinib (a Src family kinase inhibitor) significantly inhibits the growth of cells overexpressing PRKCD_pY313, with enhanced effects when combined with Adezmapimod (a p38 inhibitor) . In vivo xenograft models further validate these findings, showing that PRKCD_Y313F mutation significantly inhibits tumor progression compared to wild-type PRKCD, accompanied by decreased levels of proliferation marker Ki-67 and anti-apoptotic factor Bcl-xl, alongside increased levels of pro-apoptotic protein Bad and epithelial marker ZO1 .

What therapeutic approaches target the PRKCD_pY313 signaling pathway?

Several therapeutic approaches targeting the PRKCD_pY313 signaling pathway show promise for cancer treatment, particularly in TNBC. Dasatinib, a multi-targeted tyrosine kinase inhibitor with activity against Src family kinases, has demonstrated significant efficacy in inhibiting the growth of cancer cells overexpressing PRKCD_pY313 . This suggests that targeting Src, a downstream effector of PRKCD_pY313, represents a viable therapeutic strategy. Combination therapy approaches show enhanced efficacy, with dual inhibition of Src (using Dasatinib) and p38 (using Adezmapimod) producing stronger anti-tumor effects than either agent alone . This synergistic effect highlights the interconnected nature of these signaling pathways downstream of PRKCD_pY313. Direct targeting of PRKCD phosphorylation represents another approach, though specific inhibitors of PRKCD_pY313 are still in development. Gene therapy approaches using phospho-deficient mutants like Y313F could theoretically be explored, as these have shown anti-tumor effects in experimental models . Additionally, since PRKCD_pY313 affects mitochondrial function and ROS levels, combining targeted therapy with agents that further disrupt redox homeostasis might enhance therapeutic efficacy through synthetic lethality.

How can phosphoproteomics be used to study PRKCD_pY313 in complex biological samples?

Phosphoproteomics offers powerful approaches for studying PRKCD_pY313 in complex biological samples. For comprehensive analysis, researchers should employ a two-stage enrichment strategy: first using Superbinder resin to capture phosphorylated tyrosine (pY) peptides, followed by TiO2 columns to enrich phosphorylated serine/threonine (pS/pT) peptides . This approach allows for broader phosphoproteome coverage while maintaining sensitivity for less abundant tyrosine phosphorylation events like PRKCD_pY313. Mass spectrometry analysis should include both data-dependent acquisition for discovery and targeted methods like parallel reaction monitoring for quantitative analysis of PRKCD_pY313. For clinical samples, consider using laser capture microdissection to isolate specific cell populations from heterogeneous tissues before phosphoproteomic analysis. Data analysis should incorporate pathway enrichment, kinase activity prediction algorithms, and correlation with clinical outcomes. For validation, orthogonal techniques like Western blotting with phospho-specific antibodies should confirm key findings. This approach has been successfully applied to identify PRKCD_pY313 upregulation in breast cancer tissues compared to normal tissues, revealing its potential as a biomarker and therapeutic target .

What bioinformatic approaches can predict functional consequences of PRKCD_pY313 phosphorylation?

Bioinformatic approaches offer valuable insights into the functional consequences of PRKCD_pY313 phosphorylation. Structural analysis using homology modeling and molecular dynamics simulations can predict how Y313 phosphorylation alters PRKCD conformation, particularly given its location in the hinge region . Phosphorylation site conservation analysis across species can indicate evolutionary importance. Kinase-substrate prediction algorithms can identify potential upstream kinases responsible for Y313 phosphorylation, while phosphorylation-dependent protein-protein interaction networks can predict altered binding partners. Machine learning approaches trained on phosphoproteomic datasets can correlate PRKCD_pY313 with cellular phenotypes and patient outcomes. Pathway enrichment analysis using tools like GSEA can identify biological processes affected by PRKCD_pY313. Integration with transcriptomic data can reveal gene expression changes downstream of PRKCD_pY313 activation. Network analysis algorithms can position PRKCD_pY313 within broader signaling networks, identifying key nodes and feedback loops. These approaches complement experimental methods and can generate testable hypotheses about phosphorylation-dependent regulation of PRKCD function in normal and disease states.

How might PRKCD_pY313 serve as a biomarker in cancer diagnostics and prognostics?

PRKCD_pY313 shows considerable promise as a biomarker in cancer diagnostics and prognostics. In breast cancer, particularly TNBC, PRKCD_pY313 levels are significantly elevated compared to normal tissues . This differential expression pattern suggests potential utility as a diagnostic marker. Prognostically, high PRKCD expression correlates with poor survival outcomes, disease-specific survival, and distant metastasis-free survival in breast cancer patients . This association is particularly strong in TNBC, suggesting PRKCD_pY313 could serve as a subtype-specific prognostic indicator. For clinical implementation, immunohistochemical detection using phospho-specific antibodies could be integrated into pathology workflows. Additionally, circulating tumor cell analysis for PRKCD_pY313 might enable liquid biopsy applications for monitoring disease progression and treatment response. From a therapeutic standpoint, PRKCD_pY313 status could predict response to targeted therapies, particularly Src inhibitors like Dasatinib, as research shows PRKCD_pY313-overexpressing cells are sensitive to this treatment . Multi-marker panels incorporating PRKCD_pY313 alongside other phosphoproteins might improve diagnostic and prognostic accuracy compared to single markers.

What novel experimental models would advance our understanding of PRKCD_pY313 function?

Advancing our understanding of PRKCD_pY313 function requires development of sophisticated experimental models. CRISPR-Cas9 genome editing to generate knock-in models with phosphomimetic (Y313D/E) or phospho-deficient (Y313F) mutations would allow precise study of this modification in endogenous contexts. Patient-derived organoids maintaining the genetic and phenotypic characteristics of original tumors would provide physiologically relevant 3D models for studying PRKCD_pY313 in a tissue-like microenvironment. For temporal control, optogenetic or chemically-inducible systems to rapidly modulate PRKCD phosphorylation would enable study of acute signaling events. Single-cell phosphoproteomics approaches could reveal cell-to-cell variability in PRKCD_pY313 levels within heterogeneous tumor populations. In vivo models including conditional knock-in mice expressing phospho-variants of PRKCD would allow tissue-specific and temporal investigation of PRKCD_pY313 function. For drug development, high-throughput screening platforms with cells expressing PRKCD phospho-sensors could identify compounds that specifically modulate Y313 phosphorylation. Additionally, co-culture systems incorporating cancer cells with stromal and immune components would provide insights into how PRKCD_pY313 influences tumor-microenvironment interactions.