FHOD1 Antibody

What is FHOD1 and what are its main cellular functions?

FHOD1 is a formin family protein required for the assembly of F-actin structures, such as stress fibers. It contributes to the coordination of microtubules with actin fibers and plays a significant role in cell elongation. FHOD1 acts synergistically with ROCK1 to promote SRC-dependent non-apoptotic plasma membrane blebbing . In fibroblasts, FHOD1 is recruited to integrin clusters, resulting in actin assembly, and is essential for coordinated application of adhesive force and adhesion maturation . The targeting of FHOD1 to integrin sites depends on direct interaction with Src family kinases and is upstream of activation by Rho Kinase . Unlike other major fibroblast formins (mDia1, mDia2, or FMNL3), FHOD1 specifically localizes to integrin clusters rather than diffusely in the cytoplasm .

What applications are FHOD1 antibodies suitable for?

Based on validated research applications, FHOD1 antibodies are suitable for Western blotting (WB), immunohistochemistry on paraffin-embedded sections (IHC-P), and immunofluorescence microscopy . When using FHOD1 antibodies for Western blot analysis, researchers typically employ goat anti-mouse IgG (H&L)-HRP conjugate at 1/2500 dilution as a secondary antibody . For immunohistochemistry applications, optimal results have been achieved using formalin-fixed paraffin-embedded tissues with approximately 3 μg/ml of purified antibody . In immunofluorescence studies, FHOD1 antibodies have successfully been used to visualize both endogenous and transfected FHOD1 in conjunction with F-actin staining using phalloidin .

What is the expected molecular weight of FHOD1 in Western blot applications?

While the predicted molecular weight of FHOD1 is 127 kDa based on amino acid sequence, the observed band in Western blot applications typically appears at approximately 150 kDa . This discrepancy between predicted and observed molecular weights is important to note when analyzing Western blot results, as it may affect interpretation of protein identification. The difference could be attributed to post-translational modifications such as phosphorylation, which is known to occur at the DAD domain by Rho Kinase (ROCK) . Researchers should use appropriate positive controls and size markers to accurately identify FHOD1 bands in their experimental samples.

How is FHOD1 activated in cells?

FHOD1 activation occurs through phosphorylation of a consensus sequence in the Diaphanous Auto-regulatory Domain (DAD) by Rho Kinase (ROCK) . This phosphorylation event releases the auto-inhibitory interaction within the FHOD1 protein, promoting its actin assembly activity . Studies using phospho-specific antibodies have demonstrated that peak FHOD1 activity occurs approximately 3 to 8 minutes after cell plating, coinciding with the formation of adhesions at the cell edge . The Rho-ROCK cascade is essential for FHOD1 activity, making this protein dependent on upstream signaling from this pathway . Understanding this activation mechanism is crucial when designing experiments to study FHOD1 function in different cellular contexts.

How does FHOD1 contribute to cell adhesion and migration?

FHOD1 plays a critical role in cell adhesion and migration through multiple mechanisms. Experimental evidence from cell-spreading assays on lipid bilayers, solid substrates, and high-resolution force sensing pillar arrays demonstrates that FHOD1 knockdown significantly impairs spreading and coordinated application of adhesive force . During early cell spreading, FHOD1 is recruited to integrin clusters where it supports actin polymerization, which is essential for protrusion formation and stability . In wound scratch assays, FHOD1 knockdown cells migrate much more slowly than control cells, with approximately only one-third of the wound closed within 24 hours compared to complete closure in control cells .

At the molecular level, FHOD1 knockdown cells exhibit altered actin dynamics, with filopodia formation but failure to form coherent protrusions. These cells spread in a segmented fashion, and their protrusions frequently collapse entirely . Additionally, FHOD1-depleted cells lack strong, polarized actin filaments, and their adhesions are often limited to the periphery with a dot-like appearance rather than mature focal adhesions . These findings indicate that FHOD1 is crucial for adhesion maturation and stable protrusion formation during cell migration.

What is the significance of FHOD1 expression in cancer research?

Functional studies using lentivirus-mediated short hairpin RNA (shRNA) against FHOD1 and FHOD1-overexpression vectors have provided insights into its role in cancer progression. FHOD1 knockdown inhibits proliferation, colony formation, and migratory and invasive abilities of GC cells . Conversely, overexpression of FHOD1 promotes soft-agar colony formation and enhances migration and invasion capabilities . Co-expression analysis revealed that genes positively correlated with FHOD1 are enriched in the Gene Ontology term "extracellular matrix (ECM) structural constituent," suggesting that FHOD1 may influence cancer progression through regulation of the ECM . These findings collectively support an oncogenic role for FHOD1 in gastric cancer and potentially other cancer types.

How can I design effective FHOD1 knockdown experiments?

Designing effective FHOD1 knockdown experiments requires careful consideration of several factors. Based on published research, lentivirus-mediated short hairpin RNA (shRNA) approaches have been successfully employed to reduce FHOD1 expression in various cell types . When designing knockdown experiments, it is advisable to:

• Include appropriate controls: Use both untransfected cells and cells transfected with a non-targeting control vector to account for potential effects of the transfection procedure .

• Validate knockdown efficiency: Quantify FHOD1 mRNA expression using RT-qPCR and protein levels using Western blotting to confirm successful reduction. Published studies have achieved approximately 50% reduction in FHOD1 mRNA expression, which was sufficient to observe phenotypic effects .

• Confirm specificity through rescue experiments: Co-express siRNA-resistant human FHOD1 to restore normal phenotypes (cell area, shape, and F-actin content), which helps validate that observed effects are specifically due to FHOD1 depletion rather than off-target effects .

• Analyze multiple cellular parameters: Assess effects on cell spreading, migration (using wound scratch assays), proliferation, colony formation, and actin cytoskeletal organization to comprehensively characterize the role of FHOD1 in your cellular system .

What methods can be used to study FHOD1 activation in experimental systems?

Studying FHOD1 activation in experimental systems requires approaches that can detect the phosphorylated, active form of the protein. Several methodologies have proven effective:

• Phospho-specific antibodies: Antibodies recognizing phosphorylated consensus sequences in the DAD domain have been used to monitor FHOD1 activation. These tools can detect peak FHOD1 activity approximately 3-8 minutes after cell plating, coinciding with adhesion formation at the cell edge .

• Pharmacological manipulation: The pan-formin inhibitor smiFH2 can be used in dose-dependent studies to confirm the requirement for formin family proteins in cellular processes. This approach helps validate the specific contribution of FHOD1 to observed phenotypes .

• ROCK inhibition studies: Since FHOD1 is activated by Rho Kinase (ROCK), specific ROCK inhibitors can be employed to study the dependency of FHOD1 activation on this upstream regulator .

• Live-cell imaging: Combining fluorescently tagged FHOD1 with imaging techniques enables real-time visualization of FHOD1 recruitment to specific cellular structures such as integrin clusters. This approach has revealed that FHOD1 signal is more intense near the center of clusters but partially moves outward with actin .

How does FHOD1 interaction with the extracellular matrix (ECM) influence its function?

FHOD1's relationship with the extracellular matrix (ECM) represents an important area of research with significant implications for understanding cell-matrix interactions and potentially cancer progression. Co-expression analysis of genes correlated with FHOD1 expression has revealed enrichment in the Gene Ontology term "extracellular matrix structural constituent," suggesting a role for FHOD1 in regulating ECM integrity .

While the exact mechanisms remain under investigation, several experimental approaches have shed light on this relationship:

• Cell spreading assays on different substrates (supported lipid bilayers vs. rigid substrates) have helped distinguish FHOD1's roles in early adhesion events that occur prior to myosin contraction versus later stages of cell-matrix interaction .

• High-precision force measuring pillar arrays have demonstrated that FHOD1 is critical for coordinated application of adhesive force to the ECM, with knockdown resulting in altered inward traction stress and impaired adhesion maturation .

• Studies in gastric cancer cells suggest that FHOD1 overexpression enhances invasive capabilities, potentially through modulation of cell-ECM interactions .

These findings collectively indicate that FHOD1 serves as an important mediator between intracellular actin dynamics and cell-ECM interactions, potentially influencing tissue architecture and cancer cell invasion through regulation of both cytoskeletal organization and ECM properties.

What are optimal experimental conditions for FHOD1 antibody applications?

For optimal results when using FHOD1 antibodies in various applications, researchers should consider the following conditions based on validated protocols:

• Western Blotting: The predicted band size for FHOD1 is 127 kDa, but the observed band typically appears at approximately 150 kDa . Using goat anti-mouse IgG (H&L)-HRP conjugate at 1/2500 dilution as a secondary antibody has yielded good results in published studies . Researchers should include appropriate molecular weight markers and positive controls to accurately identify FHOD1 bands.

• Immunohistochemistry: For formalin-fixed paraffin-embedded tissues, a concentration of approximately 3 μg/ml of purified antibody has been successfully used . FHOD1 expression has been detected in various tissues, with particularly notable expression in spleen samples .

• Immunofluorescence: When co-staining with F-actin markers like phalloidin (red), FHOD1 antibodies (green) allow visualization of the relationship between FHOD1 and actin structures . DAPI can be used for nuclear counterstaining. Both transfected and endogenous FHOD1 have been detected in a periodic pattern along actin filaments, possibly related to actin bundling activity .

• Cell culture models: FHOD1 function has been successfully studied in fibroblasts and gastric cancer cell lines including HGC-27 and MKN45 . These cell lines exhibit moderate FHOD1 expression, making them suitable models for both knockdown and overexpression studies .

How can I validate the specificity of FHOD1 antibody signals?

Validating the specificity of FHOD1 antibody signals is crucial for ensuring reliable experimental results. Several approaches can be employed:

• Knockdown/knockout controls: Comparing antibody signals between wild-type cells and those with FHOD1 knockdown or knockout provides a strong validation of specificity. Reduction or elimination of signal in FHOD1-depleted samples confirms antibody specificity .

• Overexpression controls: Complementary to knockdown approaches, FHOD1 overexpression should result in enhanced antibody signal. This has been successfully demonstrated in studies using lentivirus-mediated FHOD1 overexpression, which showed approximately 5-fold higher FHOD1 expression compared to untransfected controls .

• Comparison with mRNA expression: Correlating protein detection by antibodies with mRNA expression levels measured by RT-qPCR provides additional validation of antibody specificity. In published studies, Western blotting results have supported findings from RT-qPCR analyses .

• Formin family controls: Since other formin family members (mDia1, mDia2, FMNL3) have distinct localization patterns from FHOD1, comparing their distribution patterns can help confirm the specificity of FHOD1 detection. While FHOD1 localizes to integrin clusters, these other formins typically show diffuse cytoplasmic localization .

What are the critical considerations when interpreting FHOD1 expression data in cancer studies?

When interpreting FHOD1 expression data in cancer research contexts, several important considerations should be taken into account:

• Tissue heterogeneity: Cancer tissues contain multiple cell types, including malignant cells, stromal cells, and immune infiltrates. When analyzing FHOD1 expression in bulk tissue samples, the cellular source of FHOD1 signals should be carefully determined, potentially through co-staining with cell-type specific markers .

• Correlation with clinical outcomes: While high FHOD1 expression has been associated with poor prognosis in gastric cancer patients, the prognostic significance may vary across cancer types . Proper statistical analysis, including Kaplan-Meier survival analysis with appropriate stratification (e.g., by Z-scores of FHOD1 expression), is essential for meaningful clinical correlations.

• Functional validation: Expression data should be complemented with functional studies to establish causality. Both knockdown and overexpression approaches have been used to demonstrate that FHOD1 actively contributes to cancer cell proliferation, migration, and invasion rather than merely serving as a marker .

• Pathway context: FHOD1 functions within complex signaling networks, including the Rho-ROCK cascade and interaction with Src family kinases . Interpretation of FHOD1 expression data should consider the status of these interacting pathways, as they may influence the functional consequences of altered FHOD1 expression.

• Data normalization: When comparing FHOD1 expression across samples, proper normalization to housekeeping genes (for mRNA) or loading controls (for protein) is essential. Published studies have used GAPDH normalization for Western blot quantification and β2 microglobulin for mRNA expression normalization .

What are emerging areas of FHOD1 research beyond current applications?

While significant progress has been made in understanding FHOD1's roles in cell adhesion, migration, and cancer, several promising research directions are emerging:

• Therapeutic targeting: Given FHOD1's association with poor prognosis in gastric cancer and its functional role in cancer cell proliferation and invasion, investigating specific inhibitors of FHOD1 or its activation pathway could yield potential therapeutic strategies . This might involve development of small molecule inhibitors targeting the FH2 domain or the ROCK-mediated phosphorylation sites.

• Mechanotransduction: FHOD1's involvement in coordinating actin dynamics with extracellular matrix interactions positions it as a potential mediator of mechanotransduction – the process by which cells convert mechanical stimuli into biochemical signals . Further research could explore how FHOD1 responds to substrate stiffness and mechanical forces, potentially influencing cell fate decisions.

• Immune cell function: Given FHOD1's high expression in spleen and its role in cell migration, investigating its function in immune cell dynamics, particularly in the context of tumor immunology, represents an interesting avenue for future research .

• Detailed structural studies: Although FHOD1's functional domains are known, detailed structural studies of how phosphorylation induces conformational changes and activates the protein could provide insights for structure-based drug design approaches.

• Interaction with microtubules: While FHOD1's actin-related functions are well-studied, its reported contribution to the coordination between microtubules and actin fibers warrants further investigation . Understanding this cross-cytoskeletal regulation could reveal new aspects of cell polarization and directed migration.