PDCD10 Antibody

What is PDCD10 and what cellular functions does it regulate?

PDCD10, also named CCM3 and TFAR15, belongs to the PDCD10 family. Despite its name suggesting a role in programmed cell death, recent evidence reveals it is actually an adaptor protein with multifaceted functions:

• Promotes cell proliferation and increases MAPK activity

• Modulates apoptotic pathways

• Plays important roles in cell migration

• Regulates normal structure and assembly of the Golgi complex

• Required for normal angiogenesis, vasculogenesis, and hematopoiesis during embryonic development

• Interacts with multiple molecules including serine/threonine kinases and phosphatases

Mutations in PDCD10 are one cause of cerebral cavernous malformations (CCMs), which are vascular malformations that cause seizures and cerebral hemorrhages .

What are the key specifications of commercially available PDCD10 antibodies?

PDCD10 antibodies are available in both polyclonal and monoclonal formats with the following specifications:

Polyclonal Antibodies:

Monoclonal Antibodies:

Antibodies have been validated in various cell lines including MCF-7, PC-3, Raji, Jurkat, Ramos, and U-251 cells .

What applications are PDCD10 antibodies validated for?

PDCD10 antibodies have been validated for multiple experimental applications:

The recommended antigen retrieval for IHC is with TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative .

What positive controls should I use for validating PDCD10 antibody specificity?

For proper validation of PDCD10 antibodies, the following positive controls have been experimentally verified:

• Western blot: MCF-7, PC-3, and Raji cells for polyclonal antibodies; Jurkat, Ramos, and U-251 cells for monoclonal antibodies

• Immunoprecipitation: MCF-7 cells are confirmed to yield positive results

• Immunohistochemistry: Human colon cancer tissue and human liver cancer tissue

• Gene knockdown/knockout controls: Several publications have demonstrated specificity using PDCD10 knockdown/knockout samples

It is recommended to titrate antibodies in each testing system to obtain optimal results, as sensitivity may be sample-dependent .

How should I optimize PDCD10 antibody protocols for studying its role in different cancer types?

PDCD10 has been shown to play diverse and sometimes opposing roles across different cancer types, requiring tailored experimental approaches:

For pituitary adenomas:
Research has demonstrated that PDCD10 promotes cellular proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) through the CXCR2-AKT/ERK signaling axis. When studying this cancer type:

• Use IHC to evaluate PDCD10 expression differences between non-invasive and invasive tumor samples

• Implement both mRNA (RT-PCR) and protein (WB) detection methods for comprehensive expression analysis

• Consider examining epithelial (E-cadherin) and mesenchymal markers (N-cadherin, VIM) simultaneously, as PDCD10 influences EMT

• Include scratch assays and transwell invasion assays to assess functional consequences of PDCD10 manipulation

• Monitor phosphorylation status of AKT and ERK1/2 downstream of PDCD10

For osteosarcoma:
PDCD10 is highly expressed in osteosarcoma and promotes tumor progression by inhibiting apoptosis and activating the EMT pathway:

• Examine both clinical samples and cell lines (U2OS and MG63 have been validated)

• Use shRNA for knockdown studies and cDNA overexpression for gain-of-function experiments

• Include in vivo xenograft models to confirm in vitro findings

• Analyze apoptotic markers in conjunction with PDCD10 expression

The dual roles of PDCD10 necessitate careful experimental design with appropriate positive and negative controls for each cancer context .

What are the optimal methodologies for studying PDCD10 protein-protein interactions?

PDCD10 functions as an adaptor protein that interacts with multiple binding partners. To effectively study these interactions:

Immunoprecipitation (IP):

• Use 0.5-4.0 μg of PDCD10 antibody for 1.0-3.0 mg of total protein lysate

• MCF-7 cells have been validated for successful PDCD10 IP

• Consider crosslinking to stabilize transient interactions

• Follow with western blot analysis to confirm specific binding partners

Co-immunoprecipitation for known interactions:
PDCD10 has been demonstrated to interact with:

• Protein Phosphatase 2A (PP2A) complex, including both catalytic and scaffolding subunits

• Serine/threonine kinases (STK24/25)

• CXCR2 in pituitary adenomas

When investigating these interactions, use appropriate lysis buffers (RIPA buffer with protease and phosphatase inhibitors has been validated) and include negative controls such as IgG .

Mass spectrometry approaches:
For unbiased identification of novel interaction partners, consider using immunoprecipitation followed by MALDI-TOF mass spectrometry, which has successfully identified PDCD10-interacting proteins in previous studies .

How can I effectively design experiments to study PDCD10's role in cellular signaling pathways?

PDCD10 participates in multiple signaling cascades, requiring careful experimental design:

For studying CXCR2-AKT/ERK signaling axis:

• Use RNA interference (siRNA or shRNA) for PDCD10 knockdown

• Confirm knockdown efficiency at both mRNA (qRT-PCR) and protein (western blot) levels

• Examine the phosphorylation status of downstream effectors (AKT, ERK1/2) by western blotting

• Include pathway activators (e.g., CXCL2) to confirm specificity of PDCD10's effects

• Consider rescue experiments with PDCD10 overexpression to validate findings

For investigating the role in PP2A signaling:

• Use co-immunoprecipitation to confirm PDCD10-PP2A association

• Examine the effect of PDCD10 knockdown on PP2A activity using phosphatase assays

• Include inhibitors of related pathways (Jak3, Notch, NFκB) to determine specificity

• Monitor the effect of IL-2 on PDCD10 expression and PP2A activity, as IL-2 has been shown to modulate this interaction

When studying PDCD10's dual roles, it's essential to include appropriate pathway inhibitors and activators to delineate the specific molecular mechanisms in your cellular context.

What approaches should be used to evaluate the efficacy of PDCD10 antibodies in knockout/knockdown validation studies?

Validating antibody specificity using genetic approaches is critical for ensuring result reliability:

siRNA knockdown validation:

• Transfect cells with PDCD10-specific siRNA and non-targeting control siRNA

• Confirm knockdown efficiency after 48 hours using qRT-PCR

• Use western blotting with the PDCD10 antibody to confirm protein reduction

• Include unrelated proteins (e.g., STAT3, actin) as specificity controls

• Quantify the degree of knockdown using densitometry

shRNA stable knockdown validation:

• Generate stable PDCD10 knockdown cell lines using lentiviral shRNA vectors

• Include scramble shRNA controls

• Validate knockdown at both mRNA and protein levels

• Use puromycin selection (1 mg/ml for approximately 3 weeks) to establish stable cell lines

• Test multiple shRNA constructs to identify the most effective one and rule out off-target effects

CRISPR/Cas9 knockout validation:

• For complete elimination of PDCD10 expression

• Confirm knockout using genomic PCR, sequencing, and western blotting

• Include wild-type and heterozygous controls

• Note that complete PDCD10 knockout may be embryonically lethal in some models, necessitating conditional knockout approaches

In all validation studies, the absence of PDCD10 band on western blot when using the antibody in knockout/knockdown samples provides strong evidence for antibody specificity.

What are the recommended fixation and antigen retrieval methods for PDCD10 immunohistochemistry?

Optimal detection of PDCD10 in tissue samples requires specific fixation and antigen retrieval protocols:

Fixation:

• Standard 10% neutral buffered formalin is suitable for most tissue types

• Fixation time should be optimized based on tissue thickness (typically 24-48 hours)

• Over-fixation may mask the PDCD10 epitope and require more aggressive antigen retrieval

Antigen Retrieval Methods:
Primary recommended method:

• TE buffer at pH 9.0

• Heat-induced epitope retrieval (pressure cooker or microwave)

• 20 minutes at 95-100°C

Alternative method:

• Citrate buffer at pH 6.0

• Heat-induced epitope retrieval

• 20 minutes at 95-100°C

Dilution Optimization:

• Starting dilution range: 1:50-1:500

• Titrate the antibody concentration for each tissue type

• Include positive controls (human colon cancer tissue has been validated)

• Include negative controls (antibody diluent without primary antibody)

For dual immunofluorescence studies examining PDCD10 alongside other markers, additional optimization may be required to ensure compatibility of fixation and retrieval methods for all target proteins.

How can I accurately quantify and analyze PDCD10 expression in clinical samples?

For reliable quantification of PDCD10 in patient samples:

RNA Expression Analysis:

• Use qRT-PCR with validated primers for PDCD10

• Normalize to appropriate housekeeping genes (multiple reference genes recommended)

• Consider analyzing datasets such as GSE26966 from the GEO database for comparison with normal counterparts

• Compare expression levels between invasive and non-invasive tumor samples

Protein Expression Analysis:

• Western blotting with densitometric quantification

• Use appropriate loading controls (e.g., actin)

• Consider multiplexed protein analysis for simultaneous detection of PDCD10 and related pathway proteins

Immunohistochemical Scoring:

• Use standardized scoring methods (e.g., H-score, Allred score)

• Evaluate both staining intensity and percentage of positive cells

• Consider digital image analysis for objective quantification

• Correlate expression with clinical parameters and survival data

• Compare with normal adjacent tissue when available

For prognostic studies, it's important to note that PDCD10 expression has been correlated with patient outcomes in certain cancers, such as osteosarcoma, where high expression is associated with poorer prognosis .

What are the common issues when using PDCD10 antibodies and how can they be resolved?

Researchers commonly encounter several challenges when working with PDCD10 antibodies:

High Background:

• Increase blocking time (2 hours at room temperature or overnight at 4°C)

• Use 5% BSA instead of milk for blocking in phospho-specific applications

• Increase washing steps duration and number (5-6 washes of 5-10 minutes each)

• Optimize antibody dilution (start with manufacturer's recommendation and adjust as needed)

• Pre-absorb antibody with blocking protein if non-specific binding persists

Weak or No Signal:

• Ensure proper antigen retrieval for IHC (TE buffer pH 9.0 is recommended)

• Increase antibody concentration or incubation time

• Use signal enhancement systems (HRP polymers, tyramide signal amplification)

• Check protein extraction protocol (PDCD10 is observed at 25-30 kDa)

• Verify sample preparation (fresh frozen samples may yield better results than FFPE for certain applications)

Multiple Bands in Western Blot:

• Optimize reducing conditions in sample buffer

• Use freshly prepared samples to minimize degradation

• Include protease inhibitors during sample preparation

• Consider alternative antibody clones if specific issues persist

How should PDCD10 antibodies be stored and handled to maintain optimal activity?

To preserve antibody functionality and specificity:

Storage Conditions:

• Store at -20°C as recommended by manufacturers

• Antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Stable for one year after shipment under proper storage conditions

• Aliquoting is generally unnecessary for -20°C storage according to manufacturer recommendations

• Note that some formats (20μl sizes) contain 0.1% BSA

Handling Practices:

• Avoid repeated freeze-thaw cycles

• Allow antibody to reach room temperature before opening the vial

• Centrifuge briefly before opening to collect solution at the bottom

• Use sterile technique when handling antibody solutions

• Return to -20°C promptly after use

Working Solution Preparation:

• Prepare fresh working dilutions on the day of experiment

• Dilute in appropriate buffer with 1% BSA or carrier protein

• If working solution must be stored, keep at 4°C for no more than 5-7 days

• Monitor for contamination (cloudiness or precipitation)

What controls should be included when performing PDCD10 knockdown/overexpression experiments?

For robust experimental design when manipulating PDCD10 expression:

For Knockdown Studies:

• Non-targeting siRNA/shRNA control with identical transfection/transduction conditions

• Multiple siRNA/shRNA constructs targeting different regions of PDCD10 to confirm specificity

• Validation of knockdown at both mRNA (qRT-PCR) and protein (western blot) levels

• Rescue experiments with expression of siRNA/shRNA-resistant PDCD10 construct

• Unrelated protein controls (e.g., STAT3, actin) to confirm specificity of effects

For Overexpression Studies:

• Empty vector control (e.g., pcDNA3.1)

• Tagged (e.g., Flag-tagged) and untagged versions to ensure tag doesn't interfere with function

• Expression level monitoring to avoid non-physiological overexpression artifacts

• Functional validation through known PDCD10 activities (e.g., effects on AKT/ERK phosphorylation)

For Both Approaches:

• Include wild-type untreated cells as baseline controls

• Perform time-course experiments to capture both immediate and delayed effects

• Include related pathway controls (e.g., examining PDCD10's effect on CXCR2 expression)

• Consider combinatorial approaches (knockdown + stimulation with pathway activators)

How do I design experiments to study the dual role of PDCD10 in different cellular contexts?

PDCD10 exhibits context-dependent functions that require careful experimental design:

Cellular Context Considerations:

• Use multiple cell lines representing different tissue types

• Include both cancer and normal cell counterparts when possible

• Consider primary cell cultures alongside established cell lines

• Test effects in both 2D and 3D culture systems

Molecular Pathway Analysis:

• Employ phospho-specific antibodies to monitor activation status of key signaling molecules (AKT, ERK, STAT3)

• Use inhibitors of specific pathways to determine which are essential for PDCD10's effects

• Consider inducible expression systems to study time-dependent effects

• Perform gene expression profiling to identify context-specific downstream targets

Functional Readouts:

• Include multiple functional assays (proliferation, migration, invasion, apoptosis)

• Measure effects on EMT markers as PDCD10 regulates this process in multiple cancer types

• Consider in vivo models to validate in vitro findings

• Analyze transcriptional changes using RNA-seq or targeted gene expression analysis

When studying cell death/apoptosis, use multiple complementary assays (Annexin V/7-AAD staining, caspase activation, TUNEL) to comprehensively assess PDCD10's effects .

How can PDCD10 antibodies be used to investigate its role in cerebral cavernous malformations (CCMs)?

PDCD10 mutations cause cerebral cavernous malformations with more severe symptoms than CCM1/CCM2 mutations. To investigate this:

Patient Sample Analysis:

• Use immunohistochemistry to compare PDCD10 expression in CCM lesions versus normal brain vasculature

• Correlate PDCD10 expression patterns with CCM severity and progression

• Consider laser capture microdissection to isolate specific vascular cell populations

Mechanistic Studies:

• Investigate PDCD10's interaction with VEGFR2, which is critical for vascular development

• Examine PDCD10's role in ANG-2 secretion, which is implicated in dysfunctional endothelial junctions

• Study PDCD10's regulation of the Golgi complex and its impact on vascular development

Model Systems:

• Utilize conditional knockout mouse models (complete PDCD10 knockout is embryonically lethal)

• Develop in vitro models using endothelial cells with PDCD10 mutations/knockdown

• Consider zebrafish models for high-throughput screening of potential therapeutic compounds

These approaches can provide insights into why PDCD10 mutations lead to more aggressive CCM phenotypes than other CCM genes .

What are the emerging applications of PDCD10 antibodies in cancer research and potential therapeutic development?

As PDCD10's role in cancer becomes better understood, several promising research directions are emerging:

Biomarker Development:

• Evaluate PDCD10 as a prognostic biomarker in various cancers (already validated in osteosarcoma)

• Investigate PDCD10 expression as a predictive biomarker for treatment response

• Develop companion diagnostic assays using validated PDCD10 antibodies

Therapeutic Target Validation:

• Use antibodies to track PDCD10 inhibition in preclinical studies

• Develop assays to monitor PDCD10-dependent pathways (CXCR2-AKT/ERK) in patient samples

• Investigate PDCD10's role in resistance to existing therapies

Novel Therapeutic Approaches:

• Explore small molecule inhibitors of PDCD10-protein interactions

• Investigate the potential of PDCD10-targeting antibody-drug conjugates

• Consider combination approaches targeting PDCD10 alongside conventional therapies

Personalized Medicine Applications:

• Stratify patients based on PDCD10 expression/mutation status

• Tailor treatment approaches based on PDCD10's context-specific roles

• Monitor treatment efficacy using PDCD10 and related pathway biomarkers

How can multiparametric analyses incorporating PDCD10 be designed for comprehensive understanding of its biological roles?

To fully elucidate PDCD10's complex functions, integrated approaches are necessary:

Multi-omics Integration:

• Combine proteomics, transcriptomics, and phosphoproteomics data

• Correlate PDCD10 expression with global pathway alterations

• Map PDCD10 interactome in different cellular contexts

Advanced Imaging Approaches:

• Use proximity ligation assays to validate protein-protein interactions in situ

• Implement live-cell imaging to track PDCD10 dynamics during cellular processes

• Apply super-resolution microscopy to localize PDCD10 within subcellular compartments

Systems Biology Analysis:

• Develop computational models of PDCD10-dependent signaling networks

• Predict context-specific effects of PDCD10 perturbation

• Identify key nodes for potential therapeutic targeting

Functional Genomics Screening:

• Conduct synthetic lethality screens in PDCD10-high versus PDCD10-low contexts

• Identify genetic dependencies that could be exploited therapeutically

• Use CRISPR-based approaches to systematically dissect PDCD10 function

These integrated approaches can provide a more comprehensive understanding of PDCD10's multifaceted roles across different biological contexts and disease states .

What are the recommended protocols for common PDCD10 antibody applications?

Western Blot Protocol:

• Prepare whole-tissue homogenates in RIPA buffer containing protease and phosphatase inhibitors

• Determine protein concentrations using BCA assay

• Separate 30 μg total protein by SDS-PAGE

• Transfer to nitrocellulose membrane

• Block with 5% skim milk in TBST for 1 hour at room temperature

• Incubate with PDCD10 antibody (1:500-1:6000 dilution) overnight at 4°C

• Wash 4 times with TBST, 5 minutes each

• Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Develop with ECL substrate and image

• Expected molecular weight: 25-30 kDa

Immunohistochemistry Protocol:

• Deparaffinize and rehydrate tissue sections

• Perform antigen retrieval with TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0

• Block endogenous peroxidase with 3% H₂O₂

• Block with 5% normal serum

• Incubate with PDCD10 antibody (1:50-1:500 dilution) overnight at 4°C

• Apply detection system according to manufacturer's instructions

• Counterstain, dehydrate, and mount

• PDCD10 typically shows cytoplasmic and nuclear staining patterns

Immunoprecipitation Protocol:

• Prepare cell lysates in IP buffer with protease inhibitors

• Clear lysates by centrifugation

• Pre-clear with Protein A/G beads

• Add 0.5-4.0 μg PDCD10 antibody to 1.0-3.0 mg protein lysate

• Incubate overnight at 4°C with gentle rotation

• Add Protein A/G beads and incubate for 2-4 hours

• Wash beads 3-5 times with IP buffer

• Elute proteins with 2X Laemmli buffer

• Analyze by western blotting

Where can researchers find validated PDCD10 antibodies and related resources?

Commercial Antibody Sources:

• Proteintech: Offers both polyclonal (10294-2-AP) and monoclonal (66440-1-Ig) PDCD10 antibodies with validated applications

• Thermo Fisher Scientific: Provides polyclonal PDCD10 antibody (PA5-49709)

• Several other suppliers offer antibodies against specific regions or with different host species/isotypes

Database Resources:

• UniProt ID: Q9BUL8 (Human PDCD10)

• GenBank Accession Number: BC002506

• Gene ID (NCBI): 11235

• Research Resource Identifiers (RRID): AB_2162153 (polyclonal), AB_2881810 (monoclonal)

Research Tools:

• PDCD10 knockdown vectors: Lentiviral shRNA vectors (e.g., LV-PDCD10 71721)

• PDCD10 overexpression vectors: pcDNA3.1-based constructs

• GEO datasets: GSE26966 contains PDCD10 expression data in normal versus tumor tissues

• Pathway databases: Contains information on PDCD10-related signaling networks

Most commercial antibodies come with detailed validation data, application-specific protocols, and recommended positive controls to facilitate experimental design .