RHOC Antibody

What is RHOC and what cellular functions does it regulate?

RHOC is a member of the RhoGTPase family of proteins that plays crucial roles in regulating cell physiology, including actin organization and cellular motility. Research has established RHOC as an essential factor in multiple tumor hallmarks, particularly metastasis. Studies reveal that while RHOC is indispensable for metastasis, it is not essential for normal embryogenesis in murine models . The protein regulates several cancer-related processes including epithelial-to-mesenchymal transition (EMT), migration, angiogenesis, invasion, tumor growth, and radiation response . At the molecular level, RHOC has been shown to alter the Mitogen-Activated Protein Kinase (MAPK) and Phosphoinositide 3-kinase/AKT Serine Threonine Kinase (PI3K/AKT) pathways to promote invasion, and it regulates angiogenesis via VEGF, bFGF, and interleukins in various cancer types .

What epitopes are typically targeted by RHOC antibodies for research applications?

RHOC antibodies commonly target specific amino acid sequences within the protein that serve as unique epitopes. For instance, commercially available antibodies may target regions such as amino acids 35-84, as seen with certain polyclonal antibodies . Other common epitope regions include amino acids 1-193 (full-length), 101-193, and 91-190, each providing different advantages depending on the experimental objective . When selecting an antibody, researchers should consider which functional domain of RHOC they wish to study, as different epitopes may be more suitable for detecting specific interactions or conformational states of the protein.

What species cross-reactivity can researchers expect when using RHOC antibodies?

RHOC is highly conserved across species, allowing many RHOC antibodies to demonstrate broad cross-reactivity. Based on BLAST analysis, some antibodies show 100% identity across numerous species including human, mouse, bovine, chicken, and Xenopus . This high conservation enables researchers to use the same antibody across multiple model organisms. Specifically, antibodies targeting the region between amino acids 35-84 have demonstrated reactivity with human, mouse, rat, dog, guinea pig, horse, rabbit, cow, zebrafish (Danio rerio), Drosophila melanogaster, chicken, insect cells, monkey, pig, and Xenopus laevis . This broad cross-reactivity is particularly valuable for comparative studies across evolutionary diverse models.

How can researchers optimize RHOC antibody performance in Western blotting experiments?

To optimize RHOC antibody performance in Western blotting, researchers should consider several methodological approaches:

• Sample preparation: Complete lysis of cells is essential since RHOC can localize to different cellular compartments, including the nucleus. Studies have shown that RhoC associates with WDR5 in the nucleus and regulates the expression of pluripotency genes . Therefore, ensure your lysis buffer can extract nuclear proteins effectively.

• Blocking optimization: Since many RHOC antibodies are polyclonal (like ABIN6741373), optimizing your blocking conditions is crucial to minimize background . A titration of blocking agent concentrations can help determine optimal conditions.

• Antibody concentration: Polyclonal RHOC antibodies may require different concentrations than predicted. Perform a dilution series to determine the optimal antibody concentration that maximizes specific signal while minimizing background.

• Validation controls: Include positive controls (cells known to express RHOC, such as metastatic cancer cell lines) and negative controls (RHOC-knockdown cells). Studies have validated siRNAs for RhoC that could serve as excellent negative controls .

• Detection method selection: Consider using enhanced chemiluminescence systems for higher sensitivity, particularly when detecting endogenous RHOC levels, which may be low in some cell types.

What are the key considerations when selecting between monoclonal and polyclonal RHOC antibodies?

When choosing between monoclonal and polyclonal RHOC antibodies, researchers should consider:

Polyclonal RHOC antibodies (e.g., ABIN6741373):

• Recognize multiple epitopes, providing stronger signals for proteins expressed at low levels

• Offer greater tolerance to minor protein denaturation or conformation changes

• Show broader species cross-reactivity, as evidenced by reactivity across humans, mice, and multiple other species

• May exhibit batch-to-batch variability

• Ideal for initial characterization studies and applications requiring detection of the native protein

Monoclonal RHOC antibodies:

• Target a single epitope, offering higher specificity

• Provide consistent results with minimal batch variation

• May be more suitable for distinguishing between closely related Rho family members

• Can be more sensitive to changes in protein conformation

• Preferable for quantitative analyses and specific applications requiring high reproducibility

The choice ultimately depends on the specific research application. For studying nuclear localization of RHOC or its interactions with proteins like WDR5, a highly specific monoclonal antibody might be preferred . For general detection across multiple species or in applications where protein conformation might vary, a polyclonal antibody would be more appropriate .

How can RHOC antibodies be utilized to study cancer stem cells in experimental models?

RHOC antibodies can be powerful tools for studying cancer stem cells (CSCs) through several methodological approaches:

• Co-immunofluorescence studies: RHOC antibodies can be used in conjunction with established CSC markers to identify and characterize RhoC+/Nanog+ cell populations. Research has demonstrated the existence of a subset of tumor cells marked by RhoC+/Nanog+ expression in clinical specimens . This approach allows for the visualization and quantification of potential CSC populations in both cell lines and clinical samples.

• ChIP-seq applications: RhoC has been shown to associate with WDR5 in the nucleus and regulate pluripotency genes. Chromatin immunoprecipitation sequencing (ChIP-seq) with RHOC antibodies can identify genomic regions occupied by RHOC, revealing its direct transcriptional targets. Studies have already employed this methodology to demonstrate that genes occupied by both RhoC and WDR5 are involved in pluripotency maintenance .

• Functional validation studies: After identifying RHOC-positive populations, researchers can isolate these cells and assess their stemness properties through:

  • Clonogenic assays

  • Spheroid formation assays

  • Soft agar assays

  • Anoikis resistance assays

  • Limiting dilution assays

  • Side-population analysis with Hoechst 33342

  These assays have demonstrated that RhoC regulates self-renewal ability and anoikis resistance in cancer cells .

• In vivo xenograft models: RHOC antibodies can be used to characterize tumor sections from xenografts to correlate RHOC expression with stemness markers and tumor growth characteristics. Immunofluorescent analysis of Nanog, CD49f, and ALDH in xenograft sections has shown increased expression of these markers in RHOC-overexpressing tumors .

What methodologies are most effective for studying RHOC interactions with epigenetic regulators?

To investigate RHOC interactions with epigenetic regulators such as TET2 and WDR5, researchers can employ these advanced methodological approaches:

• Co-immunoprecipitation (Co-IP): Using RHOC antibodies to pull down protein complexes, followed by Western blotting for epigenetic regulators such as TET2 or WDR5. Research has revealed that RhoC associates with WDR5 in the nucleus and regulates the expression of pluripotency genes .

• Proximity ligation assay (PLA): This technique can visualize and quantify protein-protein interactions between RHOC and epigenetic regulators within intact cells, providing spatial information about where these interactions occur subcellularly.

• ChIP-seq and Re-ChIP: Sequential ChIP experiments (first with RHOC antibody, then with antibodies against epigenetic regulators) can identify genomic regions co-occupied by both factors. Studies have already utilized ChIP-seq to reveal that the genes occupied by RhoC and WDR5 are involved in pluripotency maintenance and transcriptional processes .

• Mass spectrometry-based interactome analysis: Immunoprecipitation of RHOC followed by mass spectrometry can identify novel interacting partners within the epigenetic regulatory machinery. This approach has been used to reveal RhoC's association with WDR5 .

• Genome-wide methylation analysis: The Infinium MethylationEPIC array combined with RHOC manipulation (overexpression or knockdown) can reveal how RHOC influences global DNA methylation patterns. Research has shown that RHOC overexpression resulted in a demethylated genome via TET2 signaling .

• Reporter assays: Luciferase reporter constructs containing promoters of interest can be used to functionally validate the effect of RHOC and its epigenetic partners on gene expression. Such assays have confirmed RHOC's role in regulating pluripotency genes .

How can researchers effectively use RHOC antibodies to study nuclear localization and function of RHOC?

Studying the nuclear localization and function of RHOC requires specialized approaches:

• Subcellular fractionation and Western blotting: Careful separation of nuclear and cytoplasmic fractions followed by Western blotting with RHOC antibodies can quantify the relative distribution of RHOC between these compartments. This approach is essential as studies have identified nuclear functions of RHOC in regulating gene expression .

• Immunofluorescence microscopy with co-localization analysis: Using RHOC antibodies alongside nuclear markers (DAPI, lamin) and potential nuclear partners (WDR5, TET2) can visualize nuclear RHOC and its association with chromatin-modifying complexes .

• Chromatin association assays: Biochemical separation of chromatin-bound versus soluble nuclear proteins can determine whether RHOC directly associates with chromatin, providing insight into its mechanism of transcriptional regulation.

• Nuclear transport inhibition studies: Using specific inhibitors of nuclear import machinery while monitoring RHOC localization can help elucidate the mechanisms by which RHOC enters the nucleus.

• Site-directed mutagenesis: Creating mutations in potential nuclear localization signals (NLS) or nuclear export signals (NES) in RHOC can help identify sequences necessary for its nuclear trafficking.

• ChIP-seq with nuclear-specific controls: When performing ChIP-seq with RHOC antibodies, including controls for known nuclear and cytoplasmic proteins can help validate the specificity of nuclear RHOC binding patterns. STRING analysis of genes occupied by both RHOC and WDR5 has revealed networks involved in transcriptional regulation and developmental processes .