FAM64A Antibody

What biological functions of FAM64A should researchers consider when designing experiments with FAM64A antibodies?

FAM64A functions extend beyond its initially characterized role as a cell cycle regulator. Recent research has established FAM64A as a positive regulator of STAT3 activity through modulation of STAT3's DNA-binding activity. This regulatory function significantly impacts Th17 cell differentiation and inflammatory processes . Additionally, FAM64A has been identified as an androgen receptor-regulated feedback tumor promoter in prostate cancer, where it enhances proliferation, migration, invasion, and cell cycle progression . In head and neck squamous cell carcinoma (HNSCC), FAM64A promotes tumorigenesis through various pathways . When designing experiments, researchers should consider these diverse functions and select appropriate cellular models that express relevant signaling components and downstream targets.

What are the most reliable detection methods for FAM64A protein expression using antibodies?

Immunohistochemistry (IHC) and Western blotting represent the most reliable detection methods for FAM64A expression. For IHC, optimized protocols using FAM64A rabbit antibody (1:20 dilution) with streptavidin-biotin detection systems have demonstrated successful visualization in tissue microarrays . Assessment should incorporate both staining intensity (0-3+) and percentage of positive cells (0-4 scale) for comprehensive evaluation . Western blotting has proven effective for detecting FAM64A in multiple cancer cell lines, including prostate cancer (LNCaP, 22Rv1) and HNSCC lines, with normal cell lines (NHOK) serving as appropriate controls . Researchers should validate antibody specificity using positive controls (cancer tissues with known high expression) and negative controls (normal tissues or FAM64A-knockout samples).

How can researchers effectively use FAM64A antibodies to study its role in inflammatory diseases?

To study FAM64A's role in inflammatory diseases, researchers should utilize both in vitro and in vivo experimental approaches. In vitro, antibodies can be employed to detect FAM64A expression changes during Th17 cell differentiation or in response to inflammatory stimuli. For in vivo studies, experimental autoimmune encephalomyelitis (EAE) models in wild-type versus Fam64a-deficient mice have successfully demonstrated FAM64A's contribution to inflammatory processes . When assessing central nervous system (CNS) tissues from these models, researchers should use antibodies to evaluate both FAM64A expression and downstream inflammatory markers including IL-17A and GM-CSF . Additionally, adoptive transfer experiments with FAM64A-deficient T cells into Rag1-/- recipients have provided valuable insights into T cell-intrinsic roles of FAM64A in inflammation .

What controls are essential when using FAM64A antibodies in cancer research?

When using FAM64A antibodies in cancer research, multiple control strategies are critical:

• Tissue controls: Always include paired adjacent normal tissues (ANTs) alongside tumor samples, as demonstrated in HNSCC research where FAM64A expression was significantly higher in tumors compared to ANTs .

• Cell line controls: Include both normal cell lines (e.g., NHOK) and cancer cell lines with varying FAM64A expression levels. Studies have shown differential expression across cancer lines, with some exceptions (e.g., UM2 cell line showed lower expression) .

• Knockdown/knockout controls: siRNA-mediated knockdown of FAM64A provides excellent negative controls for antibody specificity validation. Researchers have successfully used this approach to confirm antibody specificity in prostate cancer cell lines .

• Staging controls: Include samples from different cancer stages and grades, as FAM64A expression correlates with clinical parameters including lymph node involvement, clinical stage, T stage, and differentiation status .

What methodological considerations are important when using FAM64A antibodies for phosphorylation studies?

When investigating FAM64A's relationship with phosphorylation-dependent signaling pathways, researchers should:

• Consider dual staining approaches to simultaneously detect FAM64A and phosphorylated signaling molecules (p-STAT3, p-p65) in the same tissue sections or cell populations.

• Include phosphatase inhibitors during sample preparation to preserve phosphorylation status.

• Examine both tumor and adjacent normal tissues, as studies have shown differential phosphorylation patterns between these tissue types .

• Assess both FAM64A expression and downstream phosphorylation events in response to experimental manipulations. For example, Fam64a-deficient mice exhibited decreased phosphorylation of p65 and STAT3 in colon tumors but not in adjacent normal tissues .

• Evaluate expression levels of downstream genes regulated by these phosphorylation events, such as c-Myc, Pcna, Bcl-xl, and Ccnd1, which are affected by FAM64A-mediated signaling .

How can researchers effectively use FAM64A antibodies to investigate its role in the JAK-STAT signaling pathway?

Investigating FAM64A's role in JAK-STAT signaling requires sophisticated experimental approaches:

• Phosphorylation-specific detection: Combine FAM64A antibodies with phospho-specific antibodies against STAT1, STAT2, and STAT3 to assess pathway activation. Research has demonstrated that FAM64A silencing reduces phosphorylation levels of STAT1 and STAT2 in prostate cancer cells .

• Co-immunoprecipitation assays: Use FAM64A antibodies for pull-down experiments to identify direct protein-protein interactions with JAK-STAT pathway components.

• ChIP assays: Employ chromatin immunoprecipitation with FAM64A antibodies to determine if it directly associates with chromatin at STAT-binding sites.

• Sequential immunoprecipitation: First immunoprecipitate with phospho-STAT antibodies, then probe for FAM64A to assess complex formation.

• Functional readouts: Measure expression of STAT-dependent genes (IRF9, IFI6, IFIT1-3) as functional readouts of pathway activity in the presence or absence of FAM64A .

Researchers should note that FAM64A appears to modulate type I interferon signaling through JAK-STAT pathway regulation, affecting the expression of multiple interferon-stimulated genes .

What approaches can resolve contradictory data when using FAM64A antibodies across different cancer types?

When facing contradictory results across cancer types, researchers should:

• Normalize detection methods: Standardize antibody concentrations, incubation times, and detection systems across experiments.

• Context-specific controls: Include tissue-specific controls relevant to each cancer type being studied.

• Molecular subtyping: Stratify samples according to molecular subtypes within each cancer, as FAM64A functions may vary by subtype.

• Pathway analysis: Conduct comprehensive pathway analysis to identify cancer-specific signaling networks. Transcriptome sequencing following FAM64A knockdown has revealed differential expression of 129 genes (74 upregulated, 55 downregulated) in prostate cancer, with significant enrichment in immune response and interferon signaling pathways .

• Validate with multiple antibodies: Use antibodies from different sources or targeting different epitopes to confirm findings.

• Correlative analysis: Perform correlative analysis between FAM64A expression and linearly-associated genes identified in TCGA database analysis, such as SNHG18, IFIT3, OAS1, PMSB9, OAS2, UBE2L6, IGFBP3, and ZNF48 .

How can researchers use FAM64A antibodies to explore its dual roles in immune regulation and cancer progression?

FAM64A demonstrates complex interactions between cancer progression and immune regulation. To investigate this duality:

• Immune infiltrate co-staining: Perform multiplex immunofluorescence with FAM64A antibodies alongside immune cell markers to assess spatial relationships between FAM64A-expressing cells and immune infiltrates.

• Transcriptome correlation: Compare FAM64A expression with immune-related gene signatures. Transcriptome analysis has revealed FAM64A knockdown significantly affects genes involved in virus response, type I interferon response, viral life cycle regulation, and innate immunity .

• Cytokine profiling: Measure inflammatory cytokines (TNF-α, IL-6, IL-17A, IL-17F) in serum and tissues from experimental models with varying FAM64A expression .

• Functional immune assays: Assess T cell function, particularly Th17 differentiation, in relation to FAM64A expression levels. Studies have shown that Fam64a deficiency reduces IL-17A+ and IL-17A+GM-CSF+ populations in the CNS during EAE .

• Pathway inhibitor studies: Combine FAM64A antibody detection with JAK-STAT or NF-κB pathway inhibitors to dissect the mechanistic relationships.

The table below summarizes key immune-related genes regulated by FAM64A:

What are the most reliable quantification methods for FAM64A immunohistochemistry in translational research?

For reliable quantification of FAM64A immunohistochemistry:

• Scoring system standardization: Implement a dual-parameter scoring system that accounts for both staining intensity (0-3+) and percentage of positive cells (0-4 scale, representing 0-10%, 10-25%, 26-50%, 51-75%, and 76-100%) .

• Pathologist validation: Have at least two experienced pathologists independently score samples to ensure reliability.

• Digital pathology approaches: Utilize automated image analysis software with validated algorithms for consistent quantification across samples.

• Threshold determination: Calculate final scores as the product of staining intensity and positive rate, with median value serving as the threshold for distinguishing low versus high expression groups .

• Clinical correlation: Always correlate expression levels with clinical parameters. Research has demonstrated FAM64A expression correlates with lymph node involvement, clinical stage, T stage, and differentiation status in HNSCC .

• Tissue microarray (TMA) validation: Validate findings using TMAs containing multiple tumor samples with varying grades and stages alongside normal controls .

How can researchers design experiments to investigate the potential of FAM64A as a therapeutic target using antibodies?

To investigate FAM64A as a therapeutic target:

• Neutralizing antibody studies: Test whether antibodies that block FAM64A function can reduce inflammatory responses or tumor growth in vitro and in vivo.

• Domain-specific targeting: Design experiments using antibodies targeting specific functional domains of FAM64A to determine which regions are most critical for its oncogenic and inflammatory functions.

• Combination approaches: Investigate synergistic effects of FAM64A inhibition with existing therapies, particularly those targeting STAT3 or inflammatory pathways.

• Model systems: Use multiple model systems:

  • Colitis-associated cancer (AOM/DSS) models have shown Fam64a-deficient mice develop fewer and smaller colon tumors

  • EAE models demonstrate reduced inflammatory disease in Fam64a-deficient mice

  • Xenograft models with FAM64A knockdown or overexpression can assess tumor growth kinetics

• Biomarker potential: Evaluate whether FAM64A expression levels (detected by antibodies) can serve as predictive biomarkers for response to targeted therapies.

• Downstream effector analysis: Monitor changes in downstream targets (c-Myc, Pcna, Bcl-xl, Ccnd1) and cytokine levels (TNF-α, IL-6, IL-17A, IL-17F) following FAM64A inhibition to understand mechanism of action .