RHBDF1 Antibody

What is RHBDF1 and why is it important in research?

RHBDF1 (Rhomboid Family Member 1) is a protein belonging to the Peptidase S54 family with a canonical length of 855 amino acid residues and a mass of 97.4 kDa in humans. It has critical subcellular localization in the endoplasmic reticulum (ER) and Golgi apparatus. RHBDF1 functions primarily as a regulator of ADAM17 protease, a key sheddase that processes epidermal growth factor (EGF) receptor ligands and tumor necrosis factor (TNF) . This regulatory role positions RHBDF1 as a central player in multiple cellular processes including sleep, cell survival, proliferation, migration, and inflammation. The protein is particularly important in cancer research due to its significantly elevated expression in epithelial cancers, including invasive ductal carcinoma of the breast and head and neck cancers . Its functional importance has been demonstrated through siRNA silencing experiments, which induce apoptosis in breast cancer cells and autophagy in head and neck cancer cells . Researchers investigating cancer biology, particularly signal transduction pathways involving EGFR, should consider RHBDF1 as a critical component of their studies.

What tissues and cell types typically express RHBDF1 at detectable levels?

RHBDF1 expression patterns show tissue specificity that researchers should consider when designing experiments with RHBDF1 antibodies. High expression levels have been documented in cerebellum, cerebrum, heart, skeletal muscle, placenta, pancreatic islet, and testis . In cancer research contexts, RHBDF1 protein has been readily detected in multiple human breast cancer cell lines including MDA-MB-435, MDA-MB-231, T47D, and MCF-7, with varying expression levels across these lines . Particularly noteworthy for cancer researchers is that RHBDF1 mRNA levels in MCF-7, MDA-MB-231, and T47D cells are significantly higher (approximately 3.5-, 3.1-, and 3-fold respectively) than in the immortalized but non-tumorigenic MCF-10A breast epithelial cell line . Clinical studies have found that RHBDF1 transcript is detectable in 100% of Invasive Ductal Carcinoma (IDC) stage I tumors, with expression levels nearly twice that of normal breast tissue . These expression patterns provide valuable information for researchers selecting positive control tissues or cell lines for antibody validation and experimental design.

What are the primary applications for RHBDF1 antibodies in research?

RHBDF1 antibodies serve diverse research applications with Western Blot and ELISA being the most widely used techniques . When designing experimental protocols, researchers should consider the following methodological applications:

When selecting antibodies, researchers should consider the specific epitope recognized, as this impacts the ability to detect splice variants such as RHBDF1 X6 (RHX6) . Additionally, for cancer research applications, validation in relevant cell lines (e.g., breast cancer lines for breast cancer studies) is advisable.

How can RHBDF1 antibodies be used to study its role in cancer progression?

RHBDF1 antibodies are valuable tools for investigating the critical role of this protein in cancer progression. Research has established that RHBDF1 expression is significantly elevated in clinical specimens of invasive ductal carcinoma, particularly in stage I where the relative intensity of RHBDF1 mRNA is nearly twice that of normal breast tissue . Methodologically, researchers can utilize RHBDF1 antibodies in multiple complementary approaches:

First, immunohistochemical staining of tissue microarrays containing patient samples across different cancer stages can reveal expression patterns correlating with disease progression. When performing these studies, researchers should include adjacent normal tissues as controls and quantify expression using standardized scoring systems. The significant increase in RHBDF1 expression in early-stage breast cancer suggests it may serve as an early biomarker .

Second, researchers can employ RHBDF1 antibodies in combination with phospho-specific antibodies against AKT and ERK to investigate the mechanistic link between RHBDF1 expression and activation of these critical growth signaling pathways. Silencing RHBDF1 with siRNA leads to significant down-modulation of activated AKT and ERK, suggesting these pathways are key downstream effectors . This approach requires careful validation of antibody specificity for both total and phosphorylated forms of these signaling proteins.

Third, co-immunoprecipitation experiments using RHBDF1 antibodies can identify protein interaction partners that may contribute to its oncogenic functions, such as its interaction with ADAM17/TACE and subsequent regulation of EGFR ligand processing. These studies require optimization of lysis conditions to preserve protein-protein interactions.

How can researchers effectively detect and differentiate between RHBDF1 splice variants?

Recent research has identified an important RHBDF1 gene splicing variant, RHBDF1 transcript variant X6 (RHX6), which exhibits distinct expression patterns and functions compared to canonical RHBDF1 . Methodologically, researchers face challenges in distinguishing between these variants that require careful antibody selection and experimental design.

For effective detection and differentiation, researchers should:

• Select antibodies raised against epitopes that differ between canonical RHBDF1 and RHX6. Antibodies targeting the N-terminal region may detect both variants, while those targeting regions altered by alternative splicing can differentiate between them.

• Employ RT-PCR with variant-specific primers as a complementary approach to antibody-based detection. This technique can unambiguously identify specific transcript variants and provide quantitative expression data.

• Use Western blotting with careful attention to molecular weight differences. RHX6 would have a distinct molecular weight from canonical RHBDF1.

• Consider dual-labeling immunofluorescence with antibodies specific to different regions of RHBDF1 to visualize variant-specific expression patterns within tissues or cells.

This methodological approach is particularly important given the observed inverse relationship between RHBDF1 and RHX6 expression in normal versus cancerous cells. While RHBDF1 mRNA levels are elevated in breast cancer cell lines (3-3.5 fold higher than in non-tumorigenic MCF-10A cells), RHX6 mRNA levels are markedly reduced in these same cancer cells (28.8-61.1% of MCF-10A levels) . This inverse relationship extends to clinical samples, where RHX6 expression in adjacent normal tissues significantly exceeds that in tumor tissues.

What are the methodological considerations for studying RHBDF1's regulation of ADAM17/TACE using antibodies?

RHBDF1 plays a critical role in regulating ADAM17/TACE (TNF-α converting enzyme), a sheddase that processes EGFR ligands including TGFα . Researchers investigating this regulatory relationship should consider several methodological approaches using antibodies:

First, co-immunoprecipitation experiments can demonstrate the physical interaction between RHBDF1 and TACE. These experiments have revealed that the RHBDF1 splice variant RHX6 competes with canonical RHBDF1 for binding to TACE, leading to decreased production of mature TACE . When performing co-IP experiments, researchers should optimize lysis conditions to preserve membrane protein interactions and include appropriate controls (IgG control, reciprocal IP).

Second, researchers should employ antibodies that distinguish between pro-TACE and mature TACE to assess the impact of RHBDF1 manipulation on TACE maturation. Western blotting with these antibodies can reveal how RHBDF1 or its variants influence TACE processing.

Third, immunofluorescence microscopy with antibodies against RHBDF1, TACE, and intracellular transport markers can visualize the impact of RHBDF1 on TACE trafficking through the secretory pathway. This approach is particularly valuable given that RHX6 has been shown to prevent intracellular transportation of pro-TGFα to the cell surface .

Fourth, researchers can combine antibody-based approaches with functional assays measuring TACE activity (e.g., using fluorogenic substrates) to correlate RHBDF1-TACE interaction with functional consequences.

What controls are essential when using RHBDF1 antibodies in cancer research?

When designing experiments using RHBDF1 antibodies for cancer research, incorporating appropriate controls is critical for generating reliable and interpretable data. Based on the established research, the following controls are essential:

• Expression Level Controls: Include cell lines with known RHBDF1 expression levels. MCF-10A serves as a non-tumorigenic control with baseline expression, while breast cancer cell lines such as MCF-7, MDA-MB-231, and T47D exhibit 3-3.5 fold higher expression . This comparison provides context for interpreting expression changes.

• Splice Variant Controls: Given the functional importance of the RHX6 splice variant, which shows an inverse expression pattern compared to canonical RHBDF1 in normal versus cancerous tissue , researchers should include controls that allow detection of both variants. This might involve using multiple antibodies targeting different epitopes or complementing antibody-based detection with RT-PCR.

• Knockdown/Knockout Controls: RHBDF1 siRNA-treated samples serve as specificity controls for antibody validation and provide insight into the functional consequences of RHBDF1 reduction. Research has shown that silencing RHBDF1 causes apoptosis in breast cancer MDA-MB-435 cells and autophagy in head and neck cancer 1483 cells .

• Tissue Controls: When analyzing clinical samples, adjacent normal tissue provides a critical internal control. Research has demonstrated that RHBDF1 expression is significantly elevated in IDC stage I tumors compared to normal breast tissue , making this comparison essential for contextualizing findings.

• Pathway Activation Controls: Since RHBDF1 knockdown leads to down-modulation of activated AKT and ERK , monitoring these downstream effectors serves as a functional control for RHBDF1 activity.

A systematic approach incorporating these controls enables researchers to generate robust data on RHBDF1's role in cancer biology while minimizing artifacts and misinterpretation.

How can researchers optimize RHBDF1 antibody-based detection in difficult sample types?

Optimizing RHBDF1 antibody-based detection in challenging sample types requires systematic optimization of several parameters. Researchers should consider the following methodological approaches:

For tissue samples with high lipid content (e.g., brain tissues where RHBDF1 is highly expressed ), consider extended deparaffinization steps and lipid extraction procedures before immunohistochemistry. RHBDF1's localization in the ER and Golgi necessitates careful sample preparation to preserve these membranous structures.

For samples with low RHBDF1 expression, signal amplification techniques such as tyramide signal amplification can enhance detection sensitivity. This is particularly important when studying RHX6, which shows diminished expression in cancer cells compared to normal cells .

When analyzing clinical samples that have undergone various preservation methods, optimization of antigen retrieval conditions is critical. Systematic comparison of heat-induced epitope retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0) and protease-based retrieval can identify optimal conditions for specific antibodies.

For subcellular localization studies, researchers should employ super-resolution microscopy techniques (e.g., STED, STORM) to accurately visualize RHBDF1's distribution in the ER and Golgi compartments. Co-staining with organelle markers (e.g., calnexin for ER, GM130 for Golgi) provides essential reference points.

When working with clinical samples where RNA binding motif protein-4 (RBM4) levels affect RHBDF1 splicing , researchers should consider co-staining for RBM4 to provide context for RHBDF1 variant expression patterns.

How can RHBDF1 antibodies be employed in studying epithelial-mesenchymal transition in cancer?

RHBDF1 has been implicated in epithelial-mesenchymal transition (EMT), a critical process in cancer progression. The RHBDF1 splice variant RHX6 has been shown to decrease production of EMT-related adhesion molecules when overexpressed in breast cancer cells . Researchers can employ RHBDF1 antibodies to investigate this relationship through several methodological approaches:

First, multiplex immunofluorescence staining can reveal the spatial relationship between RHBDF1 expression and EMT markers in tissue samples. Co-staining for RHBDF1 alongside E-cadherin (epithelial marker) and vimentin (mesenchymal marker) can visualize this relationship at the cellular level. Research has shown that manipulating RBM4, which controls RHX6 production, results in markedly increased E-cadherin expression and significantly decreased vimentin expression .

Second, researchers can perform sequential immunoprecipitation experiments to identify RHBDF1 interaction partners involved in EMT regulation. This approach can reveal whether RHBDF1 forms complexes with EMT transcription factors or adhesion molecules.

Third, time-course experiments combining RHBDF1 antibody staining with EMT marker detection during EMT induction (e.g., with TGFβ treatment) can elucidate the temporal relationship between RHBDF1 expression changes and EMT progression.

A comprehensive experimental design should include controls with modulated RHBDF1 expression. Overexpression of RHX6 in breast cancer cells has been shown to retard proliferation and migration , suggesting it may inhibit EMT, while canonical RHBDF1 may promote it.

What methodological approaches can reveal RHBDF1's role in AKT and ERK signaling pathways?

RHBDF1 has been implicated in modulating critical growth signaling pathways, with silencing RHBDF1 leading to significant down-modulation of activated AKT and ERK in cancer cells . Researchers investigating these signaling relationships should employ the following methodological approaches:

First, researchers should use phospho-specific antibodies against key phosphorylation sites of AKT (Ser473, Thr308) and ERK (Thr202/Tyr204) alongside total protein antibodies to assess activation status following RHBDF1 manipulation. This dual detection approach distinguishes between changes in protein levels versus activation state.

Second, time-course experiments following RHBDF1 knockdown or overexpression can reveal the kinetics of AKT/ERK pathway modulation, helping distinguish direct from indirect effects. This approach involves collecting samples at multiple time points (e.g., 6, 12, 24, 48 hours) after RHBDF1 manipulation and assessing pathway activation.

Third, researchers can employ pathway inhibitors (e.g., PI3K inhibitors for AKT pathway, MEK inhibitors for ERK pathway) in combination with RHBDF1 manipulation to determine pathway dependency. This approach can reveal whether RHBDF1's effects on cell survival, proliferation, and migration are mediated primarily through AKT, ERK, or both pathways.

Fourth, proximity ligation assays can detect close associations between RHBDF1 and signaling pathway components, potentially revealing direct interactions that mediate pathway activation.

When designing these experiments, researchers should include appropriate positive controls (e.g., growth factor stimulation for pathway activation) and negative controls (pathway inhibitors) to contextualize RHBDF1's effects.

How can researchers investigate the relationship between RHBDF1 and RBM4-mediated splicing regulation?

RHBDF1 production is subject to splicing regulation by RNA binding motif protein-4 (RBM4), which controls the balance between canonical RHBDF1 and its inhibitory splice variant RHX6 . Researchers interested in this regulatory relationship should consider several antibody-based methodological approaches:

First, researchers can perform RNA immunoprecipitation (RIP) experiments using RBM4-specific antibodies to capture RBM4-RNA complexes, followed by RT-PCR to detect RHBDF1 transcripts. This approach has demonstrated that RBM4 protein can interact with the CGGCGG motif on the RHBDF1 gene transcript . Controls should include immunoprecipitation with non-specific IgG and use of RNA molecules with mutated binding motifs.

Second, researchers can employ dual immunofluorescence staining for RBM4 and RHBDF1 proteins to assess their co-expression patterns in tissue samples. This approach is particularly valuable given that RBM4 levels in adjacent normal breast tissues are significantly higher (4.3-fold) than in tumor tissues , paralleling the pattern observed with RHX6.

Third, researchers can manipulate RBM4 expression experimentally and monitor effects on RHBDF1 and RHX6 levels using variant-specific antibodies or RT-PCR. RBM4 overexpression has been shown to substantially decrease RHBDF1 mRNA while markedly increasing RHX6 mRNA .

Fourth, chromatin immunoprecipitation (ChIP) assays using antibodies against splicing factors can identify proteins recruited to the RHBDF1 gene by RBM4, providing mechanistic insight into splicing regulation.

These approaches can reveal how the RBM4-RHBDF1 regulatory axis contributes to cancer biology, given that RBM4 overexpression decreases proliferation, migration, and colony formation in breast cancer cells .

What contradictions exist in RHBDF1 expression data across cancer types and how can researchers address them?

Current research presents some apparent contradictions in RHBDF1 expression patterns across cancer stages that warrant methodological consideration. While RHBDF1 mRNA is significantly elevated in IDC stage I tumors (nearly 2-fold higher than normal breast tissue), stages II and III show only marginal increases before expression rises again in stage IV samples . This non-linear pattern raises important questions about RHBDF1's role throughout cancer progression.

To address these contradictions, researchers should employ multiple complementary approaches:

First, researchers should use both mRNA and protein detection methods to determine whether transcript and protein levels correlate. Discrepancies could indicate post-transcriptional regulation. RT-qPCR for mRNA quantification should be paired with Western blotting and immunohistochemistry for protein detection.

Second, researchers should assess both canonical RHBDF1 and its splice variant RHX6 across cancer stages, as their relative expression might explain functional differences. The inhibitory role of RHX6 on RHBDF1 function suggests that the ratio between these variants, rather than absolute RHBDF1 levels, may be more functionally relevant.

Third, researchers should correlate RHBDF1 expression with markers of tumor heterogeneity and microenvironment composition, as these factors might explain expression variability. Multiplex immunofluorescence can simultaneously visualize RHBDF1 expression and cell type markers.

Fourth, single-cell RNA sequencing approaches can reveal cell-type specific expression patterns that might be masked in bulk tissue analysis, potentially resolving apparent contradictions.

When designing these studies, researchers should ensure adequate sample sizes across cancer stages and include matched normal-tumor samples from the same patients when possible to control for individual variability.

How can RHBDF1 antibodies facilitate research into therapeutic targeting of RHBDF1?

Given RHBDF1's critical role in epithelial cancer cell growth, with silencing leading to apoptosis or autophagy in cancer cells , it represents a promising therapeutic target. Researchers investigating therapeutic approaches targeting RHBDF1 can utilize antibodies in several methodological strategies:

First, researchers can employ antibodies to screen for compounds that modulate RHBDF1 protein levels or activity. Immunoassay-based high-throughput screening approaches can identify molecules that reduce RHBDF1 expression or disrupt its interaction with ADAM17/TACE.

Second, antibodies can assess the efficacy of gene silencing approaches in experimental models. Research has demonstrated successful delivery of siRNA to established xenograft tumors using intravenously administered histidine-lysine polymer (HKP)-encapsulated siRNA . RHBDF1 antibodies are essential for confirming target engagement in these models.

Third, researchers can develop therapeutic antibodies targeting RHBDF1 directly. Since RHBDF1 is localized in the ER and Golgi , developing antibodies that access these intracellular compartments requires specialized approaches such as antibody-drug conjugates or cell-penetrating antibodies.

Fourth, immunohistochemistry with RHBDF1 antibodies can identify patient populations likely to benefit from RHBDF1-targeted therapies based on expression levels. The finding that RHBDF1 transcript is detectable in 100% of IDC stage I tumors versus 79% in normal breast tissue suggests early-stage breast cancer as a potential therapeutic target population.