RHBDF2 Antibody

What is RHBDF2/iRhom2 and what are its main biological functions?

RHBDF2 (iRhom2) is a protein-coding gene that encodes the rhombus protease iRhom2, which functions as a cardinal regulator of EGFR signaling. RHBDF2 participates in important biological processes including growth, proliferation, and differentiation of mammalian cells . The protein plays a significant role in regulating EGFR signaling events that can promote accelerated wound healing and, when dysregulated, may trigger tumorigenesis . RHBDF2 is also critically involved in immune regulation of disease and can activate various signaling pathways including MAP3K7-dependent, EGFR, WNT, and TGF-β pathways .

What types of RHBDF2 antibodies are available for research applications?

Researchers can utilize both monoclonal and polyclonal antibodies against RHBDF2. Mouse monoclonal antibodies are available from suppliers like R&D Systems (clone not specified in data) , while rabbit monoclonal antibodies such as clone 1F17 are also commercially available . Polyclonal antibodies against RHBDF2 are offered by companies like Proteintech (23181-1-AP) . Each type has specific advantages depending on the research application - monoclonals typically offer higher specificity while polyclonals may provide stronger signal through recognition of multiple epitopes.

How is RHBDF2 protein structure related to its function?

RHBDF2 is a member of the rhomboid family but lacks proteolytic activity (hence "inactive rhomboid" or iRhom2). Its calculated molecular weight is approximately 97 kDa, comprising 856 amino acids . The protein's stability is naturally short-lived, but mutations can increase this stability and consequently enhance EGFR signaling . The protein functions as a pseudoenzyme that can stimulate secretion of specific EGF family ligands, particularly amphiregulin, independent of metalloprotease activity . This structural arrangement allows RHBDF2 to regulate EGFR signaling pathways in parallel with metalloproteases.

What are the validated applications for RHBDF2 antibodies?

RHBDF2 antibodies have been validated for multiple applications across different research platforms:

Most antibodies require optimization in each specific laboratory setting to achieve optimal results .

What is the optimal protocol for RHBDF2 immunohistochemical staining?

For optimal immunohistochemical detection of RHBDF2, antigen retrieval with TE buffer (pH 9.0) is recommended, although citrate buffer (pH 6.0) may be used as an alternative . The suggested antibody dilution ranges from 1:50 to 1:500 depending on the specific antibody and tissue type . RHBDF2 has been successfully detected in human colon cancer tissue, showing primarily cytoplasmic localization when compared to normal tissues . The staining pattern correlates with the differentiation degree of HCC tissues (r = 0.438, P = 0.005) . Always include appropriate positive controls such as human colon cancer tissue and negative controls to validate staining specificity.

How should researchers optimize Western blot protocols for RHBDF2 detection?

For Western blot detection of RHBDF2, researchers should note that the protein typically appears at approximately 97-100 kDa . A 1:1000 dilution of antibody is commonly recommended when using cell lysates such as those from HaCaT or A549 cells . Since RHBDF2 is not highly abundant in all cell types, selection of appropriate positive control samples is crucial. HCC cell lines have been shown to express varying levels of RHBDF2 , making them potential positive controls. Protein extraction methods should aim to preserve membrane proteins, as RHBDF2 is a membrane-associated protein. Use of protease inhibitors during sample preparation is essential given that RHBDF2 is naturally a short-lived protein .

What role does RHBDF2 play in cancer progression and prognosis?

RHBDF2 expression is significantly upregulated in multiple cancer types, particularly in hepatocellular carcinoma (HCC) . Overexpression of RHBDF2 predicts worse prognosis for HCC patients and is significantly associated with immune cell infiltration . RHBDF2 upregulation correlates with several clinical parameters including:

• Advanced tumor stage

• Lymph node metastasis

• TP53 mutation status

• Poorer patient outcomes

Mechanistically, RHBDF2 overexpression can activate multiple pro-oncogenic signaling pathways including EGFR, WNT, and TGF-β, which enhance cancer cell aggressiveness . In experimental models, RHBDF2 mutations increase protein stability and drive EGFR signaling, potentially triggering tumorigenesis . The protein's role in regulating EGFR signaling positions it as a potential therapeutic target in cancer research.

How does RHBDF2 influence immune cell infiltration in tumor microenvironments?

RHBDF2 expression positively correlates with immune cell infiltration in tumor microenvironments, particularly in HCC . Bioinformatic analyses using databases like TIMER have shown that RHBDF2 expression is related not only to the presence of infiltrating immune cells but also to specific immune cell markers . Gene Ontology, KEGG, and gene set enrichment analyses indicate that RHBDF2 is involved in immune signaling pathways . Interestingly, HCC patients with high RHBDF2 expression and high immune cell infiltration showed the worst clinical outcomes, suggesting that RHBDF2 may contribute to an immunosuppressive tumor microenvironment despite increased immune cell presence . This makes RHBDF2 a potential biomarker for immunotherapy response prediction.

What is the significance of RHBDF2 mutations in epithelial disorders?

RHBDF2 mutations have significant implications for epithelial disorders. Dominant mutations in RHBDF2 increase its protein stability and stimulate secretion of the EGF family ligand amphiregulin independent of metalloprotease activity . These mutations are associated with conditions such as tylosis (palmoplantar keratoderma) and epithelial cancer susceptibility . The "curly bare" (cub) mouse phenotype, characterized by wavy hair and various epithelial abnormalities, results from specific RHBDF2 mutations . These findings highlight RHBDF2's significance in maintaining epithelial homeostasis, with mutations potentially leading to accelerated wound healing in some contexts but also increasing cancer risk through sustained EGFR signaling .

How can researchers distinguish between iRhom1 and iRhom2 in experimental systems?

Distinguishing between iRhom1 (RHBDF1) and iRhom2 (RHBDF2) requires careful antibody selection and experimental design. Researchers should:

• Select antibodies specifically raised against unique regions of RHBDF2 not shared with RHBDF1

• Verify antibody specificity using positive controls (cells known to express RHBDF2) and negative controls (RHBDF2 knockout cells)

• Employ siRNA or CRISPR-based approaches to selectively silence RHBDF2 expression and confirm antibody specificity

• Consider tissue distribution differences - RHBDF2 is more predominantly expressed in immune cells while RHBDF1 shows broader tissue distribution

• Run parallel experiments with antibodies against both iRhoms to compare expression patterns

When interpreting results, researchers should be aware that these proteins share similar molecular weights and may have overlapping functions in some contexts, necessitating careful validation of antibody specificity.

What challenges exist in detecting endogenous RHBDF2 protein expression?

Detecting endogenous RHBDF2 presents several technical challenges that researchers should address:

• RHBDF2 is naturally a short-lived protein, making detection difficult without proper sample handling

• Expression levels vary significantly between tissue types and cellular contexts

• The protein's membrane association requires appropriate extraction methods to maintain structural integrity

• Post-translational modifications may affect antibody binding and protein migration patterns

• The relatively large size (~97 kDa) may require optimization of transfer conditions in Western blotting

To overcome these challenges, researchers should consider using fresh samples, optimizing protein extraction with membrane protein-specific buffers, including protease inhibitors during sample preparation, and potentially using signal enhancement techniques for detection of low-abundance RHBDF2.

How do different RHBDF2 antibody clones compare in terms of epitope recognition and performance?

Different RHBDF2 antibody clones recognize distinct epitopes, which affects their performance across applications:

Researchers should select antibodies based on their specific application needs. Monoclonal antibodies typically offer higher specificity but may be more sensitive to epitope changes, while polyclonal antibodies provide robust signal through multiple epitope recognition but potentially lower specificity. Validation in the researcher's specific experimental system is always recommended regardless of the clone selected.

What contradictory findings exist in the literature regarding RHBDF2 function?

The scientific literature contains some seemingly contradictory findings regarding RHBDF2 function:

• Pro-tumorigenic vs. Tumor-suppressive Roles: While most studies indicate RHBDF2 overexpression correlates with worse cancer prognosis through enhanced EGFR signaling , some context-dependent tumor-suppressive effects have been reported in specific cell types.

• Immune Regulation Complexity: RHBDF2 positively correlates with immune infiltration in tumors, yet high RHBDF2 expression with high immune infiltration predicts worse outcomes , suggesting complex immunomodulatory effects that may be both pro- and anti-tumorigenic depending on context.

• Metalloprotease-Dependent vs. Independent Functions: Some studies emphasize RHBDF2's role in regulating ADAM17 (a metalloprotease), while others demonstrate metalloprotease-independent functions , indicating parallel mechanisms of action.

• Tissue-Specific Effects: RHBDF2 mutations may accelerate wound healing in some tissues while promoting hyperproliferation and cancer susceptibility in others , suggesting context-dependent functions.

Researchers should carefully consider these contradictions when designing experiments and interpreting results, controlling for cell type, tissue context, and experimental conditions to reconcile these apparently conflicting findings.

What are the recommended storage and handling conditions for RHBDF2 antibodies?

Proper storage and handling of RHBDF2 antibodies is crucial for maintaining their performance:

When working with RHBDF2 antibodies, researchers should minimize freeze-thaw cycles, prepare working dilutions immediately before use, and store according to manufacturer recommendations to maintain optimal antibody performance.

How can researchers validate the specificity of RHBDF2 antibodies in their experimental systems?

To validate RHBDF2 antibody specificity, researchers should implement multiple complementary approaches:

• Positive Control Verification:

  • Use cell lines known to express RHBDF2 (e.g., HaCaT, A549, HepG2)

  • Include tissues with known RHBDF2 expression (e.g., human colon cancer tissue)

• Negative Control Testing:

  • Use RHBDF2 knockout cells generated by CRISPR/Cas9 technology

  • Employ siRNA knockdown to reduce RHBDF2 expression

  • Test in tissues/cells with minimal RHBDF2 expression

• Multiple Detection Methods:

  • Compare results across different techniques (WB, IHC, IF)

  • Use multiple antibodies targeting different RHBDF2 epitopes

  • Correlate protein detection with mRNA expression (RT-PCR)

• Peptide Competition Assay:

  • Pre-incubate antibody with immunizing peptide

  • Observe elimination of specific signal

• Molecular Weight Verification:

  • Confirm detection at expected molecular weight (~97 kDa)

  • Be aware of potential post-translational modifications affecting migration

These validation steps ensure that experimental observations genuinely reflect RHBDF2 biology rather than non-specific antibody interactions.

What are the key considerations when interpreting RHBDF2 expression data across different cancer types?

When interpreting RHBDF2 expression data across cancer types, researchers should consider several important factors:

• Baseline Expression Variations:

  • RHBDF2 expression varies significantly among normal tissues and cell types

  • Use appropriate non-malignant tissue controls for each cancer type

  • Consider RHBDF2 expression in the cell of origin for each cancer

• Correlation with Clinical Parameters:

  • Examine relationships with tumor stage, grade, and lymph node metastasis status

  • Assess association with TP53 mutation status, as this shows significant correlation

  • Evaluate prognostic value in the specific cancer context

• Immune Context Consideration:

  • RHBDF2 expression correlates with immune infiltration

  • The impact on prognosis may differ based on immune composition

  • HCC patients with high RHBDF2 and high immune infiltration show worse outcomes

• Integration with Pathway Analysis:

  • Examine correlation with EGFR, WNT, and TGF-β pathway activation

  • Consider potential cross-talk between signaling pathways

  • Evaluate context-dependent functions based on active signaling networks

• Technical Considerations:

  • Compare data across multiple platforms (RNA-seq, protein expression)

  • Be aware of potential isoform-specific expression patterns

  • Consider subcellular localization differences (cytoplasmic vs. membrane)

Comprehensive interpretation requires integration of these factors to understand the context-specific roles of RHBDF2 in different cancer types.