POPDC3 Antibody

What is POPDC3 and where is it primarily expressed?

POPDC3 (Popeye domain-containing protein 3) is a 291-amino acid transmembrane protein containing three putative transmembrane domains. It belongs to the Popeye domain-containing family, which is highly evolutionarily conserved. Under physiological conditions, POPDC3 is predominantly expressed in cardiac and skeletal muscle tissues .

Immunohistochemistry studies have detected positive POPDC3 staining in:

• Mouse heart tissue

• Human skeletal muscle tissue

• Human placenta tissue

• Human testis tissue

• Human spleen tissue

• Human lung tissue

The protein is primarily involved in signaling pathways critical for cell communication and may play roles in cell adhesion, cell motility, DNA methylation, and tumorigenesis .

What are the recommended applications for POPDC3 antibodies?

POPDC3 antibodies have been validated for multiple research applications. The recommended applications and dilutions based on experimental validation include:

For optimal antigen retrieval in IHC applications, TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) may be used as an alternative . Optimization for specific experimental conditions is often necessary to achieve ideal results.

What is the expected molecular weight of POPDC3 in Western blot analysis?

When using tagged versions of POPDC3 (such as POPDC3-CFP or POPDC3-MYC) for protein interaction studies, the molecular weight will increase accordingly based on the size of the fusion tag . For accurate assessment of POPDC3 protein expression, appropriate positive controls like COLO 320 or HEK-293 cells should be included in Western blot experiments.

How should samples be prepared for optimal detection of POPDC3?

Sample preparation is critical for successful detection of POPDC3, particularly due to its transmembrane nature. Consider these methodological approaches:

For Western blot analysis:

• For detecting POPDC3-containing complexes, cells can be lysed using 4M urea and 10% SDS without reducing agents

• Cell lysates should be sonicated and centrifuged for 30 minutes at >16,000g

• The cleared lysate should be incubated at 37°C for 30 minutes prior to Western blot analysis

For immunohistochemistry:

• Formalin-fixed, paraffin-embedded tissues are suitable for POPDC3 detection

• Antigen retrieval with TE buffer (pH 9.0) is recommended

• Optimal primary antibody dilution ranges from 1:50 to 1:500 depending on tissue type and antibody concentration

For immunoprecipitation:

• Cells should be lysed using a 1% Triton X-100 based lysis buffer supplemented with protease inhibitors

• The ProFound™ c-Myc Tag IP/co-IP Kit or Pierce HA Tag IP/Co-IP Kit have been successfully used for tagged versions of POPDC3

How does POPDC3 expression differ between normal tissues and cancer tissues?

POPDC3 shows distinct expression patterns across different cancer types compared to normal tissues, suggesting context-dependent roles:

Gastric Cancer:

• Low expression of POPDC3 was detected in 74.51% (228 of 306) of gastric cancer cases, while high expression was found in only 25.49% (78 of 306) cases

• This contrasts significantly with nontumor gastric mucosa, where POPDC3 was detected in 85.71% (72 of 84) of samples

• The differences were statistically significant (χ² = 1.010E2, P < 0.0001)

• Frequent silencing of POPDC3 in gastric cancer was associated with promoter hypermethylation

Non-Small Cell Lung Cancer (NSCLC):

• Unlike in gastric cancer, POPDC3 was significantly overexpressed in NSCLC tissues compared to adjacent non-cancerous tissues

• IHC analysis revealed notable overexpression in both lung adenocarcinoma and lung squamous cell carcinoma samples (P < 0.05)

• Elevated POPDC3 expression was significantly associated with adverse clinical outcomes and poorer survival

Head and Neck Squamous Cell Carcinoma (HNSCC):

• High POPDC3 expression was associated with radioresistance and poor prognosis in HNSCC patients

• POPDC3 was highly expressed in the post-irradiation group compared with the non-irradiation group

The subcellular localization of POPDC3 also differs between normal and cancer tissues. In normal cells, POPDC3 shows stronger membrane association, whereas in tumor cells, it exhibits predominantly cytoplasmic localization .

What role does POPDC3 play in tumor microenvironment and immune cell interactions?

Recent research has uncovered important functions of POPDC3 in modulating the tumor microenvironment (TME) and immune cell interactions:

In NSCLC, multiplex immunohistochemistry (mIHC) studies revealed:

• A significant positive correlation between elevated POPDC3 expression and enhanced infiltration of CD4+ T cells within NSCLC tissues

• No similar correlation was observed with CD8+ T cell densities

• An elevated proportion of PD-1 positive cells in NSCLC tissues exhibiting high POPDC3 expression

Functional validation in animal models demonstrated:

• Ectopic overexpression of POPDC3 in Lewis lung carcinoma (LLC) xenograft tissues in C57BL/6J mice resulted in increased CD4+ T cell infiltration

• This was accompanied by heightened PD-1 expression in the tumor microenvironment

These findings suggest a dual role for POPDC3 in immune modulation:

• Promoting T cell recruitment to the tumor site

• Potentially contributing to subsequent T cell exhaustion through increased PD-1 expression

This mechanism may explain how POPDC3-high expressing NSCLC cells could evade immune surveillance, despite triggering immune cell infiltration. The relationship between POPDC3 expression, PD-1-positive CD4+ T cell infiltration, and potential T cell exhaustion suggests POPDC3 could influence responses to immune checkpoint inhibitor therapies, though direct assessment of immunotherapy efficacy in this context requires further investigation .

How does POPDC3 influence cancer cell behavior and progression?

Functional studies have revealed multiple mechanisms through which POPDC3 influences cancer cell behavior and disease progression:

Cell Proliferation and Viability:

• In NSCLC cell lines (A549, H1299) and primary NSCLC cells, ectopic overexpression of POPDC3 significantly promoted:

  • Cell viability (measured by CCK-8 assay)

  • Colony formation capacity

  • Cell proliferation (evidenced by elevated EdU incorporation rates)

• In vivo studies confirmed that POPDC3 overexpression accelerated the growth of NSCLC xenografts in nude mice

Cell Migration and Invasion:

• POPDC3-overexpressing NSCLC cells demonstrated accelerated in vitro migration and invasion

• These effects were observed in both established cell lines and primary NSCLC cells

Epithelial-Mesenchymal Transition (EMT):

• POPDC3 overexpression promoted EMT, as evidenced by:

  • Upregulation of N-Cadherin, Vimentin, and Slug

  • Downregulation of E-Cadherin

• These EMT-related changes were detected in POPDC3-overexpressing NSCLC xenograft tissues

Apoptosis Regulation:

• POPDC3 overexpression altered the balance of pro- and anti-apoptotic proteins:

  • Increased anti-apoptotic Bcl-2 levels

  • Decreased pro-apoptotic BAX levels

In contrast to its apparent oncogenic role in NSCLC and HNSCC, in gastric cancer, reduced POPDC3 expression correlates with depth of invasion, lymph node metastasis, distant metastasis, advanced TNM stage, and poor differentiation, suggesting a potential tumor suppressor function in this context .

How do POPDC family proteins interact, and what methods can be used to study these interactions?

The POPDC family consists of three members (POPDC1/BVES, POPDC2, and POPDC3) that form complex interaction networks:

Interaction Patterns:

• POPDC1 undergoes complex formation with both POPDC2 and POPDC3

• No detectable interaction has been observed between POPDC2 and POPDC3

• These interactions occur through a helix-helix interface located at the C-terminus of the Popeye domain

• Ultra-conserved hydrophobic residues in this region are critical for mediating protein-protein interactions

Methodological Approaches for Studying POPDC Interactions:

• Co-immunoprecipitation (Co-IP):

  • Cells expressing tagged POPDC proteins can be lysed using 1% Triton X-100 based buffer with protease inhibitors

  • For POPDC3-containing complexes, cells expressing POPDC1-CFP and POPDC3-MYC can be lysed using 4M urea and 10% SDS without reducing agents

  • Commercial IP kits have been successfully used with tagged versions of POPDC proteins

• Co-transfection and Confocal Microscopy:

  • HEK293 cells can be transiently transfected with fluorescently-tagged POPDC constructs (e.g., POPDC1-CFP, POPDC2-EYFP, POPDC3-MYC)

  • Membrane localization can be visualized using membrane stains like CellBrite Red/DiD

  • This approach revealed that co-expression of POPDC1 and POPDC2 is required for proper membrane localization

• Proximity Ligation Assay (PLA):

  • PLA provides visual evidence of protein-protein interactions in situ

  • This technique has been successfully used to demonstrate heteromeric complex formation between POPDC proteins

• Bioluminescence Resonance Energy Transfer (BRET):

  • Allows quantitative measurement of protein-protein interactions in living cells

  • Has confirmed heteromeric interactions between POPDC family members

• Site-Directed Mutagenesis:

  • Mutation of specific residues in the C-terminal α-helices of the Popeye domain can disrupt protein-protein interactions

  • This approach has identified key residues required for complex formation and membrane trafficking

Understanding these interactions is crucial as they affect membrane trafficking and function of POPDC proteins, with potential implications for both normal physiology and disease states.

How can POPDC3 antibodies be optimized for multiplex immunohistochemistry studies?

Multiplex immunohistochemistry (mIHC) allows simultaneous detection of multiple proteins in a single tissue section. For successful incorporation of POPDC3 detection in multiplex panels:

Antibody Selection and Validation:

• Use POPDC3 antibodies specifically validated for IHC applications

• Perform single-plex IHC before multiplexing to:

  • Confirm specificity

  • Determine optimal dilution (typically 1:50-1:500 for POPDC3)

  • Identify optimal antigen retrieval conditions (TE buffer pH 9.0 recommended)

• When selecting multiple antibodies, ensure they are raised in different host species to avoid cross-reactivity

Panel Design Considerations:
A successful multiplex panel including POPDC3 has been demonstrated in NSCLC tissue samples, simultaneously detecting:

• PAN-CK (epithelial cell marker)

• POPDC3

• CD4 (T helper cell marker)

• CD8 (cytotoxic T cell marker)

• PD-1 (immune checkpoint marker)

Staining Protocol Optimization:

• Consider tyramide signal amplification (TSA) for increased sensitivity when detecting low-abundance proteins

• Arrange antibodies in order of decreasing sensitivity

• Optimize blocking conditions to minimize background (typically 5-10% normal serum)

• Include appropriate controls for specificity validation and autofluorescence subtraction

Quantification Methods:

• For POPDC3 expression analysis, the H-score methodology has been successfully applied

• This approach confirmed higher POPDC3 expression in NSCLC tissues compared to adjacent normal tissues

• Based on POPDC3 expression levels, tissues can be classified into "POPDC3-low expression" and "POPDC3-high expression" groups for correlation with other parameters

This approach enables assessment of POPDC3 expression in relation to tumor cells and infiltrating immune cells, providing insights into its role in the tumor microenvironment and potential implications for immunotherapy response.

How reliable is POPDC3 as a prognostic biomarker across different cancer types?

The reliability of POPDC3 as a prognostic biomarker varies significantly across different cancer types, with evidence suggesting opposing roles:

Gastric Cancer:

• Low expression of POPDC3 correlates with poor prognosis

• The 3-year survival rate for patients with low POPDC3 expression was significantly lower than for those with high expression

• Median survival difference was dramatic: 55 months (95% CI: 53.515-56.485) for high POPDC3 expression versus 23 months (95% CI: 21.201-24.799) for low expression

• For patients with low POPDC3, 1- and 3-year survival rates were 76.75% and 16.7%, compared to 88.46% and 84.6% for high expression

Non-Small Cell Lung Cancer (NSCLC):

Head and Neck Squamous Cell Carcinoma (HNSCC):

These contrasting findings highlight the context-dependent nature of POPDC3's function. The protein appears to be a reliable prognostic marker in each cancer type studied, but the direction of the association varies by cancer type, suggesting tissue-specific roles in cancer biology.

What are the key controls needed when using POPDC3 antibodies?

Proper controls are essential for ensuring reliable and interpretable results when using POPDC3 antibodies:

Positive Controls:

• For Western blot: COLO 320 cells, HEK-293 cells, and HeLa cells have been validated as positive controls for POPDC3 expression

• For IHC: Mouse heart tissue, human skeletal muscle tissue, human placenta tissue, and human testis tissue show reliable POPDC3 expression

• Primary cardiac and skeletal muscle tissues are optimal physiological positive controls due to high endogenous POPDC3 expression

Negative Controls:

• Antibody specificity can be validated using POPDC3 knockdown cells (siRNA or CRISPR/Cas9)

• For IHC, omission of primary antibody while maintaining all other staining steps

• Competitive peptide blocking using the immunogenic peptide to confirm specificity

Validation of Specificity:

• Cross-reactivity with other POPDC family members should be assessed, particularly POPDC1 and POPDC2

• When using overexpression systems, confirm that POPDC3 overexpression doesn't affect expression levels of POPDC1 and POPDC2

• For tagged versions, verify that the tag doesn't interfere with protein localization or function

Internal Controls for Clinical Samples:

• When analyzing tumor samples, including adjacent normal tissue provides an internal reference

• For prognostic studies, categorizing samples into "POPDC3-high" and "POPDC3-low" groups requires consistent scoring methods (e.g., H-score methodology)

Implementation of these controls enhances confidence in experimental results and facilitates accurate interpretation of POPDC3 expression and function across different experimental contexts.

What are the challenges in interpreting POPDC3 expression in different cancer contexts?

Researchers face several challenges when interpreting POPDC3 expression data across different cancer contexts:

Contrasting Prognostic Implications:

• POPDC3 shows opposite prognostic associations in different cancer types:

  • Reduced expression correlates with poor prognosis in gastric cancer

  • Increased expression correlates with poor prognosis in NSCLC and HNSCC

• These contradictions suggest context-dependent functions requiring careful interpretation

Technical Considerations:

• The observed molecular weight (60 kDa) differs significantly from the calculated molecular weight (34 kDa)

• Possible post-translational modifications may affect antibody recognition

• Membrane proteins like POPDC3 require specific sample preparation to avoid aggregation or degradation

Expression Level Interpretation:

• Relatively weak expression in certain NSCLC tissues and cells raises questions about physiological relevance

• Elevated POPDC3 expression observed in lentivirus-transfected NSCLC cells may not accurately reflect conditions in actual tumors

• Absolute quantification of POPDC3 in a larger cohort of primary tumor samples is needed for context

Subcellular Localization Variations:

• POPDC3 localization differs between normal and malignant tissues

• Predominantly membrane-associated in normal cells

• Mainly cytoplasmic in tumor cells

• These localization differences may reflect altered function or protein interactions

Interaction with Immune Microenvironment:

• High POPDC3 expression correlates with increased CD4+ T cell infiltration and PD-1 expression

• This represents a complex relationship where increased immune cell infiltration (typically beneficial) is accompanied by increased immune checkpoint expression (potentially immunosuppressive)

To address these challenges, researchers should:

• Consider tissue-specific contexts when interpreting POPDC3 expression data

• Validate findings across multiple technical approaches

• Assess both expression levels and subcellular localization

• Incorporate functional studies to understand the biological implications of altered POPDC3 expression

What novel approaches can be used to study POPDC3 function in cancer?

Several innovative approaches can advance our understanding of POPDC3's role in cancer:

CRISPR/Cas9-Mediated Genome Editing:

• Generate POPDC3 knockout cell lines to assess loss-of-function effects

• Create knock-in models with tagged endogenous POPDC3 to study localization without overexpression artifacts

• Introduce specific mutations identified in cancer patients to evaluate functional consequences

Patient-Derived Organoids (PDOs):

• Establish 3D organoid cultures from patient tumors with varying POPDC3 expression levels

• Use these models to study:

  • Drug sensitivity correlations with POPDC3 expression

  • Effects of modulating POPDC3 expression on organoid growth and invasion

  • Impact on tumor-immune cell interactions in co-culture systems

Single-Cell Analysis:

• Apply single-cell RNA sequencing to understand heterogeneity of POPDC3 expression within tumors

• Combine with spatial transcriptomics to correlate POPDC3 expression with location in the tumor microenvironment

• Integrate with proteomic data to identify co-expression patterns

Advanced Imaging Techniques:

• Use live-cell imaging with fluorescently tagged POPDC3 to track dynamic changes in localization

• Apply super-resolution microscopy to precisely determine subcellular localization

• Implement Förster resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) to study protein-protein interactions in real time

Integrative Multi-Omics Analysis:

• Correlate POPDC3 expression with:

  • DNA methylation patterns (particularly relevant given evidence of promoter hypermethylation in gastric cancer)

  • Chromatin accessibility

  • Proteome and phosphoproteome profiles

Syngeneic Mouse Models with Immune Component:

• Established models in C57BL/6J mice with Lewis lung carcinoma (LLC) have successfully demonstrated POPDC3's impact on immune cell infiltration

• These models can be expanded to:

  • Test immunotherapy efficacy in relation to POPDC3 expression

  • Evaluate combination therapies targeting both POPDC3 and immune checkpoints

  • Study dynamics of CD4+ T cell recruitment and functional status