FAM174A Antibody

What is FAM174A and what are its known biological functions?

FAM174A, also known as Transmembrane protein 157 (TMEM157) or Hepatitis C virus NS5A-transactivated protein 6, is a protein whose function remains largely uncharacterized. The protein contains a DUF1180 domain with unknown function . Recent research has revealed its significance primarily through fusion gene studies where FAM174A-WWC1 fusion has demonstrated oncogenic potential in colorectal cancer. The fusion protein formed by FAM174A preserves its DUF1180 domain, which appears to contribute to cellular transformation, enhanced migration, and invasive capabilities when fused with the C-terminal portion of WWC1 .

What validation methods should be employed to confirm FAM174A antibody specificity?

To verify FAM174A antibody specificity, researchers should implement a comprehensive validation approach. First, western blotting should be performed using the antibody (recommended dilution: 0.04-0.4 μg/mL) against samples with known FAM174A expression . Enhanced validation through recombinant expression is critical, as demonstrated with available antibodies like HPA019539 . The Human Protein Atlas project has extensively characterized such antibodies through protein arrays testing 364 human recombinant protein fragments to ensure minimal cross-reactivity . For validation in complex samples, researchers should consider using positive and negative control tissues and evaluating signal presence in expected subcellular compartments as FAM174A demonstrates specific localization patterns.

What are the optimal sample preparation techniques for FAM174A immunodetection?

For optimal FAM174A immunodetection, sample preparation varies by technique. For immunohistochemistry (recommended dilution 1:50-1:200), formalin-fixed paraffin-embedded tissues should undergo appropriate antigen retrieval . For immunoblotting applications (0.04-0.4 μg/mL), protein extraction should be performed using buffers that effectively solubilize membrane proteins, as FAM174A contains transmembrane domains . When studying subcellular localization via immunofluorescence (0.25-2 μg/mL), fractional protein extraction techniques can be informative, as demonstrated in studies of FAM174A-WWC1 that revealed distinct cytoskeletal localization patterns . For complete protein extraction, particularly when investigating potential fusion proteins, subcellular fractionation should be employed to examine cytoplasmic, nuclear, and cytoskeletal distributions separately .

What controls are essential when using FAM174A antibody in immunohistochemistry?

When conducting immunohistochemistry with FAM174A antibody, several controls are indispensable. Positive controls should include tissues known to express FAM174A based on Human Protein Atlas data, which has characterized expression across 44 normal human tissues . Negative controls should include both technical controls (primary antibody omission) and biological controls (tissues known not to express the target). For studies examining potential fusion proteins like FAM174A-WWC1, it's crucial to incorporate antibodies targeting different epitopes of both fusion partners to distinguish between the full-length proteins and fusion products . Additionally, when investigating pathological conditions, matched normal-tumor tissue pairs should be used to contextualize altered expression patterns, as demonstrated in EOCRC research where differential expression patterns provided insights into oncogenic mechanisms .

How can FAM174A antibody be used to investigate the FAM174A-WWC1 fusion protein in cancer research?

To investigate the FAM174A-WWC1 fusion protein in cancer research, researchers should employ a multi-faceted approach. First, domain-specific antibodies targeting the DUF1180 domain of FAM174A should be used alongside antibodies recognizing the C-terminal regions of WWC1 to specifically detect the fusion protein . Immunoprecipitation followed by mass spectrometry can confirm the presence of the fusion protein in clinical samples. For functional studies, as demonstrated in previous research, immunocytochemistry should be performed to analyze subcellular localization, which revealed that FAM174A-WWC1 predominantly localizes to cytoskeletal regions and lamellipodia, with particularly strong expression at cellular edges . Signal intensity analysis along cell profiles can quantitatively demonstrate this distribution pattern. Western blotting of subcellular fractions has confirmed that the fusion protein is primarily detected in the cytoskeletal fraction, informing its potential role in focal adhesion and extracellular matrix interactions .

What methodological approaches should be used to study the role of FAM174A in cellular transformation and invasion?

Studying FAM174A's role in cellular transformation and invasion requires sophisticated methodological approaches. Based on established research protocols, investigators should first establish stable cell lines expressing FAM174A or relevant fusion constructs like FAM174A-WWC1 using lentiviral transduction in non-transformed cell lines such as HEK293 and NIH3T3 . Cellular transformation can be assessed through morphological analysis, as previous research has documented a shift from stellate to round and polygonal morphology upon FAM174A-WWC1 expression . For invasion studies, both two-dimensional basement membrane matrix assays and three-dimensional spheroid invasion assays should be implemented. In the 3D approach, cells are grown as cyst-like forms and seeded with invasion matrix to monitor dissemination into surrounding matrigels . Complementary migration assays in two dimensions can further characterize motility alterations. These functional tests should be coupled with molecular analyses examining epithelial-mesenchymal transition markers, as FAM174A-WWC1 expression has been shown to decrease E-cadherin and augment N-cadherin expression .

How can researchers distinguish between wild-type FAM174A and fusion proteins in experimental samples?

Distinguishing between wild-type FAM174A and fusion proteins requires strategic experimental design. Researchers should employ RT-PCR with primers spanning potential fusion breakpoints to specifically amplify fusion transcripts . For protein detection, antibodies targeting unique epitopes created at fusion junctions provide the highest specificity. Alternatively, using antibodies against regions preserved in the fusion protein but targeting epitopes from different fusion partners (e.g., FAM174A N-terminus and WWC1 C-terminus) can confirm co-localization indicative of fusion . Size discrimination via Western blotting is particularly informative, as fusion proteins typically exhibit altered molecular weights compared to wild-type proteins (e.g., FAM174A-WWC1 appears at approximately 100-123 kDa) . For functional discrimination, researchers should assess downstream signaling alterations, as FAM174A-WWC1 uniquely increases both total YAP1 and phosphorylated YAP1 in the nucleus, unlike wild-type FAM174A . RNA sequencing comparison between cells expressing wild-type versus fusion proteins can identify distinctly affected pathways, such as the focal adhesion and ECM receptor pathways significantly inhibited by FAM174A-WWC1 .

What analytical approaches should be employed when interpreting conflicting FAM174A localization patterns across different cell types?

When confronting conflicting FAM174A localization patterns across cell types, researchers should implement a systematic analytical approach. First, high-resolution confocal microscopy with Z-stack analysis should be employed to generate three-dimensional localization maps, as demonstrated in studies showing FAM174A-WWC1 concentrates in lamellipodia and cytoskeletal regions . Quantitative signal intensity analysis along defined cell profiles can objectively measure distribution patterns and resolve apparent discrepancies . Complementary biochemical fractionation (separating nuclear, cytoplasmic, membrane, and cytoskeletal components) provides independent verification of localization, as shown in studies where fusion proteins exhibited differential compartmentalization compared to wild-type proteins . Context-dependent localization should be evaluated by examining cells under various conditions, including growth factor stimulation, matrix attachment variations, and cell density differences. Co-localization studies with markers for specific subcellular structures can clarify ambiguous patterns, particularly when examining potential translocation events. When integrating conflicting reports, researchers should consider technical variables (fixation methods, antibody epitopes) and biological variables (cell-type specific interaction partners) that might explain divergent observations.

How should researchers design experiments to investigate the impact of FAM174A in tumorsphere formation and stem-like properties?

Designing experiments to investigate FAM174A's impact on tumorsphere formation requires a carefully controlled approach. First, establish stable cell lines with inducible expression systems for FAM174A or fusion constructs like FAM174A-WWC1 to enable temporal control over expression . Seed equal numbers of control and FAM174A-expressing cells in ultra-low attachment plates using serum-free media supplemented with essential growth factors to promote sphere formation. Quantify sphere formation efficiency by counting the number and measuring the size of tumorspheres after 7-14 days . For serial propagation assays, dissociate primary spheres and re-seed at equal densities to assess self-renewal capacity over multiple generations . Complement functional assays with molecular characterization of stemness markers (ALDH activity, CD133, CD44) through flow cytometry. For mechanistic insights, analyze the relationship between FAM174A expression and known stem cell regulating pathways (e.g., YAP1 signaling), as previous studies demonstrated that FAM174A-WWC1 increases both total YAP1 and phosphorylated YAP1 levels in the nucleus . Include appropriate positive controls (established oncogenes known to enhance stemness) and negative controls to contextualize findings.

What experimental approaches can resolve the relationship between FAM174A and the focal adhesion and ECM receptor pathways?

To resolve the relationship between FAM174A and focal adhesion/ECM receptor pathways, researchers should implement multi-level experimental approaches. At the transcriptome level, RNA sequencing comparing control cells with FAM174A-expressing or FAM174A fusion-expressing cells can identify significantly altered genes within these pathways, as previous research has demonstrated that FAM174A-WWC1 significantly inhibits focal adhesion and ECM receptor pathways . At the protein level, researchers should analyze key components of these pathways using phospho-specific antibodies to detect activation states of focal adhesion kinase (FAK), paxillin, and other adhesion complex proteins. Co-immunoprecipitation experiments can identify direct protein interactions between FAM174A and focal adhesion components. Live-cell imaging using fluorescently tagged focal adhesion proteins can visualize dynamic changes in response to FAM174A manipulation. Functionally, adhesion strength assays and traction force microscopy can quantify the mechanical consequences of FAM174A expression. The relationship to invasiveness should be evaluated through 3D matrix degradation assays using fluorescently labeled matrix proteins to visualize proteolytic activity at invasion sites . Finally, rescue experiments reintroducing key focal adhesion proteins in FAM174A-overexpressing backgrounds can establish causality in the observed phenotypes.

What are the critical parameters for optimizing FAM174A antibody usage in different experimental techniques?

Optimizing FAM174A antibody usage requires careful attention to technique-specific parameters. For immunohistochemistry, critical factors include antigen retrieval method, antibody dilution (1:50-1:200 recommended), incubation time, and detection system sensitivity . For immunoblotting, optimization should focus on protein extraction method (considering FAM174A's membrane association), transfer conditions (for optimal transfer of proteins across the molecular weight spectrum), blocking agent selection, antibody concentration (0.04-0.4 μg/mL recommended), and exposure time . For immunofluorescence applications, fixation method significantly impacts epitope accessibility, with recommended antibody concentrations of 0.25-2 μg/mL . When investigating potential fusion proteins, researchers must optimize lysis conditions to ensure complete extraction from relevant cellular compartments, as FAM174A-WWC1 has been shown to concentrate in cytoskeletal fractions . For all applications, temperature and duration of antibody incubation should be systematically tested. Validation experiments should include peptide competition assays using the immunogen sequence (RRNRKTRRYGVLDTNIENMELTPLEQDDEDDDNTLFDANHPRR) to confirm specificity . Signal amplification methods should be calibrated to prevent saturation while maintaining sensitivity for detecting low-abundance targets.

What control experiments are essential when investigating the transformative capacity of FAM174A fusion proteins?

When investigating the transformative capacity of FAM174A fusion proteins, comprehensive control experiments are essential. Expression controls should include wild-type FAM174A, the fusion partner (e.g., WWC1) alone, and truncated versions of each to distinguish fusion-specific effects from individual protein contributions . Functional rescue experiments should test whether reintroducing wild-type domains can reverse fusion-induced phenotypes. For cellular transformation assays, positive controls such as known oncogenes (e.g., KRAS mutants) provide benchmarks for comparison. When assessing tumorsphere formation capacity, researchers should include progressive dilution series to quantify tumor-initiating cell frequency . For migration and invasion assays, control experiments must include both vector-only controls and cells expressing individual fusion components to isolate fusion-specific effects . When analyzing downstream signaling (e.g., YAP pathway activation), parallel experiments with pathway inhibitors can confirm causality . For in vivo studies, researchers should include both negative controls (vector-only cells) and positive controls (established oncogene-expressing cells) alongside cells expressing the FAM174A fusion being studied. Statistical rigor requires multiple biological replicates and appropriate statistical tests to account for variation inherent in transformation assays.

How should researchers interpret differences in results when using antibodies targeting different epitopes of FAM174A?

When interpreting differences in results from antibodies targeting different FAM174A epitopes, researchers must consider several factors. Epitope accessibility varies by technique and sample preparation, particularly for transmembrane proteins like FAM174A . Researchers should map the epitope locations relative to functional domains (e.g., DUF1180) and potential post-translational modification sites to understand potential masking effects . In fusion protein contexts, certain epitopes may be lost at breakpoints, while others remain accessible, creating discrepancies in detection patterns. For example, N-terminal FAM174A antibodies would detect both wild-type and the FAM174A-WWC1 fusion, while C-terminal antibodies would only detect the wild-type protein if the fusion occurs mid-protein . When conflicting results emerge, researchers should implement confirmatory approaches using alternative detection methods such as mass spectrometry or mRNA analysis. Epitope-specific antibodies can serve as useful tools to map protein interactions if certain regions become inaccessible upon binding to partners. When publishing discrepant findings, researchers should explicitly report the epitope locations of antibodies used, fixation conditions, and detection methods to facilitate interpretation across studies.

How can FAM174A antibody be used to analyze the relationship between FAM174A-WWC1 fusion and YAP1 signaling in cancer progression?

To analyze the relationship between FAM174A-WWC1 fusion and YAP1 signaling in cancer progression, researchers should employ a comprehensive strategy using FAM174A antibodies. Immunoprecipitation studies can detect physical interactions between the fusion protein and components of the Hippo-YAP pathway . Western blotting of subcellular fractions (cytoplasmic versus nuclear) can quantify changes in YAP1 localization induced by the fusion protein, as research has demonstrated that FAM174A-WWC1 increases both total YAP1 and phosphorylated YAP1 in the nucleus . Immunofluorescence co-localization studies can visualize FAM174A-WWC1 and YAP1 distribution patterns in single cells. For functional analysis, researchers should combine FAM174A antibody-based detection with reporter assays measuring YAP-dependent transcriptional activity (using TEAD response elements). ChIP-seq experiments can map genome-wide changes in YAP1 binding patterns in the presence of the fusion protein. Proximity ligation assays can detect direct interactions between the fusion protein and YAP1 or upstream regulators. To establish causality, researchers should employ genetic approaches (YAP1 knockdown/knockout) in cells expressing the fusion protein to determine if YAP1 is required for the observed oncogenic phenotypes . Time-course analyses can reveal the sequence of molecular events following fusion protein expression.

What analytical methods should be used to quantify and compare FAM174A expression levels in normal versus cancer tissues?

Quantifying and comparing FAM174A expression levels in normal versus cancer tissues requires robust analytical methods. For immunohistochemical analysis, tissue microarrays containing matched normal-tumor pairs should be employed with standardized staining protocols . Automated digital image analysis using validated algorithms can provide objective quantification of staining intensity and percentage of positive cells across multiple tissue cores. H-score calculation (intensity × percentage) offers a comprehensive measurement for statistical comparison. For protein quantification, Western blotting with appropriate loading controls and standard curves generated using recombinant protein can provide absolute quantification . RNA-level quantification through RT-qPCR with validated reference genes or RNA-seq with appropriate normalization allows transcript-level comparison. When analyzing FAM174A in potential fusion contexts, breakpoint-spanning primers in RT-PCR can specifically quantify fusion transcripts separately from wild-type . For comprehensive characterization, researchers should perform correlation analyses between FAM174A expression levels and clinicopathological features (tumor stage, grade, patient outcomes). When comparing across studies, researchers must consider technical variables (antibody clones, detection methods) and implement meta-analysis approaches to integrate diverse datasets. Single-cell analyses can reveal heterogeneity in expression that might be obscured in bulk tissue analyses.

How can researchers distinguish between direct effects of FAM174A alterations and secondary adaptations in experimental models?

Distinguishing direct effects of FAM174A alterations from secondary adaptations requires sophisticated experimental design. Inducible expression systems provide temporal control, allowing researchers to identify immediate versus delayed responses to FAM174A manipulation . Time-course experiments analyzing both transcript and protein changes can establish the sequence of events following FAM174A alteration. For fusion proteins like FAM174A-WWC1, domain mutation studies can identify which specific regions are necessary for observed phenotypes . Acute versus chronic expression models can separate initial direct effects from compensatory adaptations. Pharmacological rescue experiments using inhibitors of downstream pathways can determine whether observed phenotypes require continued signaling or represent irreversible changes. For mechanistic insights, researchers should combine FAM174A antibody-based detection with transcriptomic and proteomic profiling at multiple time points after FAM174A manipulation. Chromatin immunoprecipitation experiments can identify direct transcriptional targets versus indirect effects. Single-cell analyses can reveal population heterogeneity in response to FAM174A alterations, potentially identifying resistant subpopulations. When analyzing tumor models, researchers should compare early versus late passage cells/tumors to identify drift in phenotypes that might indicate secondary adaptations rather than direct FAM174A effects.

What are the common technical challenges when detecting FAM174A-WWC1 fusion proteins, and how can they be addressed?

Detecting FAM174A-WWC1 fusion proteins presents several technical challenges. First, fusion-specific antibodies recognizing the unique junction sequence are ideal but often unavailable, requiring researchers to use antibodies against preserved domains in each fusion partner . RNA-level detection using RT-PCR with primers spanning the fusion junction provides high specificity but may miss protein-level alterations . For low-abundance fusion proteins, enrichment through immunoprecipitation prior to Western blotting can enhance detection sensitivity. Subcellular fractionation is critical, as FAM174A-WWC1 has been shown to concentrate in the cytoskeletal fraction, which may be incompletely extracted using standard lysis buffers . When analyzing clinical samples, tissue heterogeneity can dilute fusion protein signals, necessitating microdissection techniques. For morphological analysis of cells expressing the fusion protein, researchers should implement standardized criteria for classifying changes from stellate to round/polygonal shapes . In multiplexed immunofluorescence, spectral overlap between fluorophores can confound co-localization analysis, requiring appropriate controls and spectral unmixing. For functional validation, researchers should confirm fusion transcript expression via RT-PCR with fusion-spanning primers before attributing phenotypes to the fusion protein .

What methodological refinements are needed when analyzing FAM174A in tissues with high autofluorescence or background staining?

Analyzing FAM174A in challenging tissues requires methodological refinements. For tissues with high autofluorescence (e.g., liver, brain), researchers should implement spectral imaging with unmixing algorithms to distinguish specific signal from autofluorescence . Tissue-specific blocking protocols (using appropriate sera and elevated protein concentrations) can reduce background in immunohistochemistry applications. For immunofluorescence in such tissues, photobleaching prior to antibody application, followed by autofluorescence quenchers (e.g., Sudan Black B), can dramatically improve signal-to-noise ratios. When analyzing formalin-fixed paraffin-embedded tissues, extended antigen retrieval protocols may be necessary to expose FAM174A epitopes . For quantitative analysis, background subtraction algorithms using unstained or isotype control-stained serial sections provide reference values. Multiplexed approaches combining FAM174A antibody with cell-type specific markers can help distinguish specific staining in heterogeneous tissues. Signal amplification methods such as tyramide signal amplification should be calibrated to prevent artifactual signal enhancement. For tissues with endogenous peroxidase activity, dual peroxidase blocking steps are essential before applying HRP-conjugated detection systems. When using polyclonal antibodies like HPA019539, absorption against tissue homogenates from negative control samples can reduce non-specific binding .

How should researchers approach data integration when combining FAM174A expression analysis with genomic and functional studies?

Researchers approaching data integration when combining FAM174A expression analysis with genomic and functional studies should implement a multi-level analytical framework. For integrating protein expression data (from antibody-based methods) with genomic alterations, researchers should use matched samples to directly correlate FAM174A protein levels with copy number variations, mutations, or fusion events . Computational approaches such as weighted gene co-expression network analysis (WGCNA) can identify modules of genes that correlate with FAM174A expression. When analyzing fusion events like FAM174A-WWC1, researchers should correlate the presence of the fusion (detected by breakpoint-spanning PCR) with altered signaling pathways (e.g., YAP/TAZ activity) . Multivariate statistical approaches can help distinguish FAM174A-specific effects from confounding variables. For functional integration, researchers should implement in vitro phenotypic assays (invasion, tumorsphere formation) and correlate outcomes with molecular profiles . Pathway enrichment analysis of RNA sequencing data can identify biological processes altered by FAM174A manipulation, as demonstrated in studies showing that FAM174A-WWC1 significantly affects focal adhesion and ECM receptor pathways . Network analysis combining protein-protein interaction data with expression changes can identify key nodes mediating FAM174A effects. When integrating clinical data, machine learning approaches can identify patterns associating FAM174A alterations with patient outcomes, treatment responses, or clinical features.

What methodological controls are necessary when studying the effects of FAM174A on epithelial-mesenchymal transition markers?

When studying FAM174A effects on epithelial-mesenchymal transition (EMT) markers, rigorous methodological controls are essential. Positive controls should include cell lines with established EMT phenotypes (e.g., TGF-β-treated epithelial cells) to benchmark FAM174A-induced changes . Multiple EMT markers should be assessed simultaneously, including epithelial markers (E-cadherin, claudins, occludin) and mesenchymal markers (N-cadherin, vimentin, fibronectin), as FAM174A-WWC1 has been shown to decrease E-cadherin and augment N-cadherin expression . Time-course experiments are critical to distinguish transient from stable EMT programs. For protein-level validation of EMT marker changes, researchers should use multiple antibody clones targeting different epitopes of each marker. When analyzing EMT in 3D culture systems, researchers should implement live-cell imaging to track morphological changes over time, complemented by endpoint immunostaining . EMT transcription factor activity (SNAIL, SLUG, ZEB1/2, TWIST) should be assessed to determine the molecular drivers behind observed marker changes. For functional validation, migration and invasion assays should be performed in parallel with marker analysis to confirm phenotypic EMT . Genetic rescue experiments reintroducing epithelial markers (e.g., E-cadherin) can establish causality between marker loss and observed phenotypes. Epigenetic analysis of EMT marker promoters can provide mechanistic insights into stable versus transient expression changes.