RHBDD1 Antibody

What is RHBDD1 and what cellular functions does it perform?

RHBDD1 is a member of the evolutionarily conserved Rhomboid family of polytopic membrane serine proteases involved in growth and development processes. It functions primarily as a protease that cleaves specific target proteins, including BIK (a proapoptotic member of the Bcl-2 family) and proTGFα. RHBDD1 is highly expressed in testis tissue and plays important roles in modulating apoptotic activity and potentially in mammalian spermatogenesis . The protein is localized in both the plasma membrane (particularly in filopodial-like projections) and the cytoplasm, suggesting diverse cellular functions depending on its subcellular location . Recent evidence indicates that RHBDD1 may have significant functions in cancer progression through its involvement in growth factor signaling pathways .

What is the molecular structure and characteristics of RHBDD1?

RHBDD1 is a 315 amino acid protein with a calculated molecular weight of approximately 36 kDa, which corresponds to its observed molecular weight in experimental conditions . It contains a characteristic rhomboid domain that confers its proteolytic activity. As a membrane-associated protein, RHBDD1 has several transmembrane domains that anchor it to cellular membranes. The protein possesses a catalytic site essential for its proteolytic function, as demonstrated by studies showing that catalytically inactive RHBDD1 cannot trigger proTGFα cleavage and secretion . The protein is encoded by the RHBDD1 gene (Gene ID: 84236 in humans and 76867 in mice) and has several aliases including RHBDL4 and RRP4 .

What are the standard applications for RHBDD1 antibodies?

RHBDD1 antibodies are commonly employed in several key laboratory techniques:

• Western Blot (WB): Used at dilutions typically ranging from 1:1000 to 1:6000 to detect RHBDD1 protein expression levels in tissue and cell lysates .

• Immunohistochemistry (IHC): Applied at dilutions of 1:50 to 1:500 to visualize the distribution and localization of RHBDD1 in tissue sections .

• ELISA: Utilized to quantify RHBDD1 protein levels in biological samples .

• Immunofluorescence: Employed to determine subcellular localization, particularly when coupled with epitope tags such as FLAG .

These antibodies have demonstrated reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across species .

Which tissue types express RHBDD1 and how can this be detected?

RHBDD1 exhibits varied expression patterns across different tissues. It is highly expressed in testis tissues, suggesting an important role in reproductive biology and potentially in spermatogenesis . Western blot analyses have detected RHBDD1 expression in multiple tissues and cell types including mouse kidney tissue, mouse testis tissue, HEK-293 cells, and PC-3 cells .

In colorectal cancer research, RHBDD1 has been found to be significantly upregulated in cancer samples compared to adjacent normal tissues, as demonstrated through tissue microarray analysis . This differential expression pattern makes RHBDD1 a potential biomarker for certain cancer types. Immunohistochemical staining with anti-RHBDD1 antibodies is an effective method for visualizing tissue-specific expression patterns, with positive staining detected in human testis tissue using established protocols with either TE buffer (pH 9.0) or citrate buffer (pH 6.0) for antigen retrieval .

How does RHBDD1 contribute to cancer progression and what methodologies best demonstrate this relationship?

RHBDD1 has been implicated in cancer progression, particularly in colorectal cancer (CRC), through several mechanisms. Research has shown that RHBDD1 expression is significantly upregulated in CRC samples compared to adjacent normal tissues, and this upregulation is associated with poor prognosis . The oncogenic role of RHBDD1 can be investigated through multiple methodological approaches:

• Gene inactivation studies: RHBDD1 inactivation through somatic cell knock-in methods has been shown to inhibit tumor cell growth in proliferation assays and colony formation assays. Mutation of RHBDD1 results in significant decreases in colony size and quantity in soft agar assays, demonstrating reduced anchorage-independent growth .

• Xenograft models: In vivo studies utilizing tumor xenografts have demonstrated that RHBDD1-mutant cells produce significantly smaller tumors compared to wild-type cells, with decreased expression of proliferation markers such as Ki67 and PCNA .

• Pathway analysis: RHBDD1 interacts with and cleaves proTGFα, leading to its secretion and subsequent activation of the EGFR signaling pathway, which is crucial for cancer cell growth and survival .

Researchers should employ a combination of these approaches to comprehensively evaluate RHBDD1's role in cancer, including both in vitro cellular models and in vivo animal studies to validate findings across experimental systems.

What are the optimal protocols for validating RHBDD1 antibody specificity?

Validating antibody specificity is crucial for obtaining reliable research results. For RHBDD1 antibodies, the following validation protocol is recommended:

• Western blot analysis with positive and negative controls:

  • Use known RHBDD1-expressing tissues/cells (e.g., testis tissue, HEK-293 cells) as positive controls .

  • Include RHBDD1 knockout or knockdown samples as negative controls to confirm specificity.

  • Verify that the detected band appears at the expected molecular weight of 36 kDa .

• Peptide competition assay:

  • Pre-incubate the antibody with a blocking peptide (e.g., PEP-0974 for antibody PA5-20860) .

  • Compare results with and without blocking peptide to confirm specific binding.

• Cross-validation with multiple antibodies:

  • Use different antibodies targeting distinct epitopes of RHBDD1.

  • Compare staining patterns to ensure consistency across antibodies.

• Genetic validation:

  • Utilize RHBDD1 gene knockout or knockdown models.

  • For example, researchers have validated anti-RHBDD1 monoclonal antibodies by examining their reactivity in RHBDD1-inactivated cell lines created using somatic cell knock-in methods .

• Immunoprecipitation followed by mass spectrometry:

  • Confirm that the immunoprecipitated protein is indeed RHBDD1.

These comprehensive validation steps ensure that experimental observations attributed to RHBDD1 are not artifacts of non-specific antibody binding.

How can researchers effectively study RHBDD1's protease activity?

Investigating RHBDD1's protease activity requires specialized methodologies that can detect substrate cleavage and product formation. Based on research findings, the following approaches are recommended:

• Cell-based secretion assays:

  • Incorporate epitope tags (e.g., FLAG) into potential substrate proteins like proTGFα between the signal peptide and the functional domain .

  • Co-express the tagged substrate with wild-type or catalytically inactive RHBDD1.

  • Analyze culture medium for cleaved substrate products using immunoblotting.

  • Include metalloprotease inhibitors (e.g., BB94) to distinguish RHBDD1-mediated cleavage from other proteases .

• Dose-dependency experiments:

  • Vary the expression levels of RHBDD1 to establish a correlation between protease concentration and substrate cleavage efficiency .

• Substrate specificity analysis:

  • Compare multiple potential substrates to determine cleavage site preferences.

  • Use site-directed mutagenesis of substrate cleavage sites to map exact cleavage positions.

• ELISA-based detection methods:

  • Quantify secreted substrate products (e.g., TGFα) in the presence of active or inactive RHBDD1 .

• In vitro reconstitution assays:

  • Purify recombinant RHBDD1 and substrate proteins.

  • Conduct controlled cleavage reactions in defined buffer conditions.

When designing these experiments, researchers should include appropriate controls, particularly catalytically inactive RHBDD1 mutants and other rhomboid family members like RHBDL2, to confirm specificity of the observed proteolytic activities .

What techniques are most effective for studying RHBDD1's protein-protein interactions?

Understanding RHBDD1's interaction network is crucial for elucidating its cellular functions. Based on research findings, these methodological approaches are recommended:

• Affinity purification coupled with mass spectrometry:

  • Tag endogenous RHBDD1 by knock-in of epitopes (e.g., FLAG and SBP) to the C-terminus .

  • Perform affinity purification using anti-epitope antibodies.

  • Analyze purified protein complexes by SDS-PAGE followed by silver staining.

  • Identify interacting proteins by mass spectrometric analysis, ranking candidates by protein scores .

• Bidirectional co-immunoprecipitation:

  • Express tagged versions of RHBDD1 and potential interacting partners.

  • Perform co-immunoprecipitation experiments in both directions (pull-down RHBDD1 to detect partner, and pull-down partner to detect RHBDD1).

  • This approach has successfully verified interactions between RHBDD1 and proTGFα .

• Functional validation of interactions:

  • Design experiments to test whether the identified interactions have functional consequences.

  • For example, assess whether RHBDD1 can cleave an interacting protein like proTGFα in secretion assays .

• Subcellular co-localization studies:

  • Use immunofluorescence microscopy to determine whether RHBDD1 and its interacting partners co-localize in specific cellular compartments.

  • This approach confirmed RHBDD1's localization in both plasma membrane and cytoplasm of HCT116 cells .

• Domain mapping:

  • Create truncated versions of RHBDD1 to identify which domains are responsible for specific protein interactions.

These combined approaches provide comprehensive insights into RHBDD1's protein interaction network and the functional significance of these interactions.

What methods are appropriate for investigating RHBDD1's role in apoptosis regulation?

RHBDD1 has been implicated in apoptosis regulation through its interaction with pro-apoptotic proteins like BIK. To study this function, researchers should consider these methodological approaches:

• Gain and loss-of-function experiments:

  • Overexpress RHBDD1 or suppress it using RNAi in appropriate cell lines.

  • These complementary approaches have demonstrated that RHBDD1 overexpression reduces BIK-mediated apoptosis, while suppression enhances it .

• Apoptosis assays:

  • Measure apoptotic markers after modulating RHBDD1 expression.

  • Use flow cytometry with Annexin V/PI staining to quantify apoptotic cells.

  • Assess caspase activation and PARP cleavage by immunoblotting.

• BIK cleavage assays:

  • Co-express tagged versions of RHBDD1 and BIK.

  • Monitor BIK processing and degradation in the presence of wild-type or catalytically inactive RHBDD1.

• Cell survival studies in physiological contexts:

  • For example, when studying RHBDD1's role in spermatogenesis, researchers suppressed RHBDD1 expression by RNAi in GC-1 cells (a spermatogonia cell line) and found that these cells lost their ability to survive and differentiate in mouse seminiferous tubules .

• Pathway analysis:

  • Investigate how RHBDD1-mediated regulation of apoptosis intersects with other cellular pathways.

  • Explore whether RHBDD1's role in apoptosis is connected to its function in growth factor signaling.

These methodologies provide complementary approaches to understand RHBDD1's complex role in apoptotic regulation across different cellular contexts.

How can researchers optimize RHBDD1 antibody use in different applications?

Optimizing antibody use requires application-specific considerations. For RHBDD1 antibodies, the following technical recommendations apply:

For Western Blotting (WB):

• Use dilutions between 1:1000 and 1:6000, with exact dilution dependent on the specific antibody and sample type .

• K562 cell lysate serves as an effective positive control for many RHBDD1 antibodies .

• Include appropriate molecular weight markers to verify the expected 36 kDa band size .

• For detecting mutant RHBDD1 proteins that may be degraded through the proteasome pathway, consider treating samples with proteasome inhibitors (e.g., Velcade, MG132) prior to analysis .

For Immunohistochemistry (IHC):

• Use dilutions between 1:50 and 1:500 .

• Optimize antigen retrieval methods; both TE buffer (pH 9.0) and citrate buffer (pH 6.0) have been successfully used with RHBDD1 antibodies .

• Human testis tissue serves as a reliable positive control for IHC applications .

• Include appropriate negative controls such as isotype control antibodies or tissues known to lack RHBDD1 expression.

For Immunofluorescence:

• When studying subcellular localization, epitope tagging approaches (e.g., FLAG-tagging RHBDD1) have proven effective .

• Use subcellular markers to confirm localization patterns, such as plasma membrane and cytoplasmic markers.

Each application should be individually titrated to determine optimal conditions for specific experimental systems and antibody preparations.

What are the common challenges in detecting endogenous RHBDD1 and how can they be overcome?

Detecting endogenous RHBDD1 can present several challenges that researchers should anticipate and address:

• Low expression levels: RHBDD1 may be expressed at low levels in certain tissues or cell types.

  • Solution: Use sensitive detection methods such as enhanced chemiluminescence for WB or amplification steps in IHC protocols.

  • Consider sample enrichment through subcellular fractionation since RHBDD1 is present in both membrane and cytoplasmic fractions .

• Protein degradation: Mutant RHBDD1 can be rapidly degraded through the ubiquitin/proteasome pathway.

  • Solution: Treat samples with proteasome inhibitors (Velcade, MG132) to stabilize the protein for detection .

• Antibody specificity: Non-specific binding can complicate interpretation of results.

  • Solution: Validate antibody specificity using RHBDD1 knockout or knockdown samples as negative controls.

  • Use epitope-tagged knock-in approaches for studying endogenous RHBDD1, which have successfully been employed in HCT116 cells .

• Cross-reactivity with other rhomboid family members: RHBDD1 shares sequence homology with other rhomboid proteins.

  • Solution: Ensure the antibody has been validated against other family members, particularly RHBDL2, which has been used as a comparison in functional studies .

• Tissue fixation effects: Overfixation can mask RHBDD1 epitopes in tissue samples.

  • Solution: Optimize fixation protocols and antigen retrieval methods for IHC applications.

By anticipating these challenges and implementing appropriate technical solutions, researchers can improve the reliability of endogenous RHBDD1 detection.

How can RHBDD1 antibodies be utilized in cancer research?

RHBDD1 antibodies serve as valuable tools in cancer research, particularly given the protein's upregulation in colorectal cancer and its role in tumor growth . The following applications are especially relevant:

• Prognostic biomarker studies:

  • RHBDD1 upregulation has been associated with poor prognosis in colorectal cancer .

  • Antibodies can be used in tissue microarrays to evaluate RHBDD1 expression across large patient cohorts.

  • Correlate expression levels with clinical outcomes to assess prognostic value.

• Therapeutic target validation:

  • Antibodies can help validate RHBDD1 as a potential therapeutic target.

  • Use in combination with RHBDD1 inhibition strategies to monitor target engagement and biological effects.

• Pathway analysis:

  • Investigate RHBDD1's role in the EGFR signaling pathway through its cleavage of proTGFα .

  • Examine how RHBDD1 inhibition affects downstream signaling events and cancer cell phenotypes.

• Translational research:

  • Compare RHBDD1 expression between normal tissues, primary tumors, and metastatic lesions.

  • Evaluate RHBDD1 as a companion diagnostic for potential targeted therapies.

• Mechanistic studies:

  • Investigate how RHBDD1-mediated protease activity contributes to tumor cell growth and invasion.

  • Use antibodies in combination with gene editing techniques to study the consequences of RHBDD1 deletion or mutation in cancer models .

These applications highlight the versatility of RHBDD1 antibodies in advancing our understanding of cancer biology and potentially developing new therapeutic strategies.

What experimental approaches can elucidate RHBDD1's role in spermatogenesis?

Given RHBDD1's high expression in testis and its potential role in spermatogenesis , researchers can employ these methodological approaches:

• Expression profiling during spermatogenesis:

  • Use RHBDD1 antibodies for immunohistochemical analysis of testis sections at different developmental stages.

  • Correlate expression patterns with specific cell types and maturation stages.

• Cell line models:

  • Utilize spermatogonia cell lines like GC-1 that can differentiate into spermatids.

  • Previous research has shown that suppression of RHBDD1 by RNAi in GC-1 cells causes them to lose the ability to survive and differentiate in mouse seminiferous tubules .

• Ex vivo organ culture systems:

  • Manipulate RHBDD1 expression in testicular explants.

  • Evaluate effects on spermatocyte survival, differentiation, and maturation.

• Conditional knockout models:

  • Generate testis-specific RHBDD1 knockout mice.

  • Assess fertility, sperm count, morphology, and function.

• Identification of testis-specific substrates:

  • Utilize co-immunoprecipitation and mass spectrometry to identify RHBDD1-interacting proteins in testicular cells.

  • Validate potential substrates through cleavage assays similar to those used for proTGFα .

• Comparative analysis with other reproductive disorders:

  • Examine RHBDD1 expression and function in models of male infertility or reproductive pathologies.

These approaches can provide comprehensive insights into RHBDD1's function in male reproductive biology and potentially inform treatments for certain forms of male infertility.