PNMA5 Antibody

What is PNMA5 and why is it important in cancer research?

PNMA5 (Paraneoplastic Ma antigen 5) is a confirmed tumor onconeural antigen that has been identified as a female fertility factor. Research indicates that PNMA5 is significantly overexpressed in epithelial ovarian cancer tissues compared to benign ovarian tumors and normal ovarian tissues. Its expression levels correlate with FIGO stage and histological grade, suggesting PNMA5 plays a crucial role in the occurrence and development of epithelial ovarian cancer. Studies have demonstrated that PNMA5 is essential for meiosis in mouse oocytes and may serve as a marker of poor prognosis in multiple cancer types . This makes PNMA5 antibodies important tools for detecting expression patterns in various tissues and investigating underlying molecular mechanisms in cancer research.

What types of PNMA5 antibodies are commonly used in research?

Research commonly employs several types of PNMA5 antibodies:

• Rabbit polyclonal anti-PNMA5 antibodies (such as those from Sigma) - These are widely used for immunohistochemistry and western blot analyses due to their high sensitivity and specificity for total PNMA5 protein detection .

• Phosphorylation-specific antibodies (such as rabbit anti-p-PNMA5) - These specialized antibodies detect only the phosphorylated form of PNMA5, which is critical for understanding PNMA5 activation in signaling pathways. These have been used in studies examining the relationship between PNMA5 phosphorylation and upstream regulators like Src kinase .

The choice between these antibody types depends on the specific research question and whether the focus is on total protein expression or phosphorylation status.

What are typical dilution ratios for PNMA5 antibodies in different applications?

Based on published research protocols, optimal dilution ratios for PNMA5 antibodies vary by application:

• For immunohistochemistry: Rabbit polyclonal anti-PNMA5 antibodies are typically used according to manufacturer recommendations, with optimization for specific tissue types.

• For western blot analysis: Primary PNMA5 antibodies are commonly diluted in 5% skim milk powder solution and incubated with membranes at 4°C overnight .

• For immunofluorescence: Phospho-PNMA5 (p-PNMA5) antibodies have been successfully used at a 1:1,000 dilution ratio followed by incubation with fluorescent secondary antibodies (such as Cy2-conjugated donkey anti-rabbit IgG) .

Researchers should always perform antibody titration experiments to determine optimal concentrations for their specific experimental conditions and sample types.

How can PNMA5 antibodies be used to investigate signaling pathways in cancer cells?

PNMA5 antibodies provide valuable tools for elucidating the role of PNMA5 in cancer-related signaling pathways. Research has revealed that PNMA5 phosphorylation is positively regulated by Src activity and that phosphorylated PNMA5 can modulate the downstream glycogen synthase kinase-3β (GSK-3β) signaling pathway .

A comprehensive approach involves:

This methodological approach has demonstrated that Src acts upstream of PNMA5, and phosphorylated PNMA5 positively regulates GSK-3β phosphorylation, which has implications for cancer cell proliferation and invasion .

What controls should be included when using PNMA5 antibodies in expression studies?

Robust PNMA5 antibody studies require comprehensive controls:

• Positive tissue controls: Epithelial ovarian cancer tissues have been confirmed to express high levels of PNMA5 (84.0% positive expression rate) and serve as excellent positive controls .

• Negative/low expression controls: Normal ovarian tissues and benign ovarian tumors show significantly lower PNMA5 expression (35.0% and 36.7% positive expression rates, respectively) and can serve as comparative controls .

• Cell line controls: The ovarian cancer cell line HO8910 (high PNMA5 expression) and normal ovarian cell line Moody (lower PNMA5 expression) provide appropriate cellular models for antibody validation .

• PNMA5 knockdown controls: siRNA-mediated PNMA5 knockdown samples should be included to confirm antibody specificity.

• Loading controls: For western blot analyses, GAPDH antibody should be used as a loading control to normalize protein expression levels .

The table below summarizes PNMA5 expression in different tissue types based on immunohistochemical analysis:

ᵃ compared with benign ovarian tumor tissue
ᵇ compared with normal healthy ovarian tissue
ᶜ compared with ovarian cancer tissue

How can PNMA5 antibodies be used in functional studies following gene knockdown?

PNMA5 antibodies are essential tools for validating knockdown efficiency and investigating functional consequences in gene silencing experiments. A methodological approach includes:

• Transfection validation: Following siRNA transfection targeting PNMA5, western blot analysis using anti-PNMA5 antibodies confirms knockdown efficiency at the protein level .

• Phosphorylation status assessment: Using phospho-specific PNMA5 antibodies to determine how knockdown affects not just protein levels but also activation status.

• Downstream pathway analysis: Employing antibodies against downstream targets like p-GSK-3β to evaluate pathway disruption following PNMA5 knockdown .

• Correlation with functional changes: Connecting antibody-detected molecular changes with functional outcomes like proliferation (using CCK-8 assay), cell cycle distribution (PI staining/flow cytometry), and apoptosis (Annexin V-FITC/PI double staining) .

Research demonstrates that PNMA5 knockdown in HO8910 cells not only reduces PNMA5 protein levels but also affects downstream GSK-3β phosphorylation, correlating with inhibited proliferation, increased G1 phase percentage, and elevated early apoptosis rates .

What are the optimal fixation and antigen retrieval methods for PNMA5 immunohistochemistry?

Successful PNMA5 immunohistochemistry requires careful sample preparation:

• Fixation: Research protocols recommend 4% paraformaldehyde fixation for tissue specimens, followed by standard dehydration procedures, paraffin embedding, and sectioning .

• Antigen retrieval: While specific antigen retrieval methods for PNMA5 aren't explicitly detailed in the provided studies, common approaches for nuclear/cytoplasmic antigens typically include:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Controlled heating (95-100°C) for 15-20 minutes followed by cooling

• Blocking: Effective blocking of non-specific binding sites using 5% skim milk powder solution before antibody incubation .

• Incubation conditions: Overnight incubation at 4°C with primary PNMA5 antibodies yields optimal staining results .

The scoring system for PNMA5 expression should assess both percentage of positive cells (0-3 points) and staining intensity (0-3 points), with combined scores determining negative/positive classification .

What are common issues encountered when using PNMA5 antibodies in western blot analysis?

Researchers may encounter several challenges when using PNMA5 antibodies in western blot applications:

• Non-specific binding: PNMA5 belongs to the PNMA family with some sequence homology among members. Use blocking with 5% skim milk powder solution and careful antibody dilution optimization to minimize cross-reactivity .

• Phosphorylation detection: When studying both total and phosphorylated PNMA5, consider:

  • Running duplicate blots (one for each antibody)

  • Using phosphatase inhibitors during sample preparation

  • Employing membrane stripping protocols carefully to prevent protein loss

• Transfer efficiency: PNMA5 requires semi-dry transfer methods to PVDF membranes for optimal detection .

• Signal detection optimization: Enhanced chemiluminescence (ECL) techniques using ECL advance reagents are recommended for PNMA5 detection. Signal quantification should be performed using appropriate software (such as Image J) .

• Validation of results: Always compare PNMA5 expression between cancer cell lines (like HO8910) and normal cell lines (like Moody) as experimental controls .

How can PNMA5 antibodies be combined with other techniques to validate expression findings?

A multi-technique approach strengthens PNMA5 expression analysis:

• Immunohistochemistry + Western blot: Combining these techniques allows correlation between tissue localization patterns and protein size/quantity validation .

• Immunofluorescence + Subcellular fractionation: These complementary approaches confirm the subcellular distribution of PNMA5, which has been observed in both cytoplasm and nucleus .

• siRNA knockdown validation: Confirming antibody specificity by demonstrating reduced signal following PNMA5 siRNA treatment. The following siRNA target sites have been effectively used:

• Correlation with mRNA expression: Validating protein-level findings using RT-PCR or RNA-seq to confirm transcriptional changes mirror protein expression patterns.

• Functional assays: Connecting antibody-detected expression with biological outcomes through proliferation, migration, invasion, and apoptosis assays .

How should PNMA5 expression patterns be interpreted in different cancer types?

Interpretation of PNMA5 expression requires careful consideration of tissue type and pattern:

• Expression localization: PNMA5 has been observed in both cytoplasm and nucleus of cells, with potential functional implications for each localization pattern .

• Tissue-specific expression: Current research shows significantly higher PNMA5 expression in epithelial ovarian cancer (84.0% positive rate) compared to benign ovarian tumors (36.7%) and normal ovarian tissues (35.0%) .

• Correlation with clinical parameters: PNMA5 expression correlates with FIGO stage and histological grade in ovarian cancer, suggesting prognostic value .

• Expression in cell lines: Consistently higher expression in cancer cell lines (HO8910) compared to normal ovarian cell lines (Moody) reinforces its potential role in oncogenesis .

• Phosphorylation status: Phosphorylated PNMA5 levels provide insights into activation status and relationship with upstream kinases like Src .

When interpreting results, consider both the percentage of positive cells and staining intensity, using standardized scoring systems to classify samples as negative (−/+) or positive (++/+++) .

What does PNMA5 phosphorylation indicate about cancer cell signaling pathways?

PNMA5 phosphorylation status provides critical insights into cancer cell signaling dynamics:

• Src kinase relationship: Research demonstrates that PNMA5 phosphorylation is positively regulated by Src activity. When Src inhibitors are applied, phosphorylated PNMA5 levels decrease dramatically while total PNMA5 remains unchanged, confirming Src acts upstream of PNMA5 .

• GSK-3β pathway connection: Phosphorylated PNMA5 positively regulates the GSK-3β pathway. Following PNMA5 knockdown, phosphorylated GSK-3β is significantly downregulated while total GSK-3β remains unchanged .

• Functional implications: This signaling cascade (Src → phosphorylated PNMA5 → phosphorylated GSK-3β) appears to influence multiple cancer hallmarks:

  • Cell proliferation

  • Cytoskeletal organization (F-actin polymerization)

  • Cell migration and invasion

  • Apoptosis resistance

  • Mitochondrial distribution

These findings suggest PNMA5 phosphorylation serves as a critical signaling node connecting upstream oncogenic kinases to downstream effectors controlling cancer cell behavior.

How can PNMA5 antibody results contribute to biomarker development in clinical research?

PNMA5 antibody findings provide valuable insights for biomarker development:

• Diagnostic potential: The significant difference in PNMA5 expression between ovarian cancer tissues (84.0% positive) and benign/normal tissues (~36% positive) suggests diagnostic utility .

• Prognostic implications: Correlation with FIGO stage and histological grade indicates PNMA5 could serve as a prognostic biomarker, similar to its role in colon cancer .

• Therapeutic target validation: PNMA5 knockdown studies demonstrate multiple anti-cancer effects:

  • Inhibited proliferation

  • Blocked cell cycle progression

  • Promoted early apoptosis

  • Decreased migration and invasion

  • Disrupted microfilament organization

  • Altered mitochondrial distribution

• Companion diagnostic development: PNMA5 phosphorylation status might predict responsiveness to Src inhibitors or other targeted therapies affecting this pathway.

• Multi-marker panels: Combining PNMA5 with other biomarkers could enhance diagnostic or prognostic accuracy for ovarian cancer.

Research suggests that PNMA5 "plays a vital role in the occurrence and development of EOC and is a potential biomarker for this disease" , making PNMA5 antibodies valuable tools for translational cancer research.