RHBDL2 Antibody

What is RHBDL2 and what are its known functional characteristics?

RHBDL2 (Rhomboid-like 2) is an evolutionarily conserved intramembrane serine protease that localizes to the plasma membrane in mammalian cells. It encodes the protein 'rhomboid like 2' in humans, also known as RRP2, rhomboid-related protein 2, and rhomboid (veinlet, Drosophila)-like 2. Structurally, the protein has a molecular mass of approximately 34 kilodaltons .

RHBDL2 functions primarily as a protease that catalyzes the cleavage of cell surface transmembrane proteins, resulting in the release of their ectodomains. This process, known as ectodomain shedding, plays critical roles in various signaling pathways. Most notably, RHBDL2 can cleave EGF ligands, thereby triggering EGFR activation, which is implicated in cellular proliferation and survival mechanisms . Unlike other proteases that may have redundant functions, RHBDL2 appears to have specific substrate repertoires that are resistant to cleavage by other proteases such as ADAMs (A Disintegrin And Metalloproteases) .

What experimental applications are RHBDL2 antibodies most commonly utilized for?

RHBDL2 antibodies are employed in multiple experimental techniques crucial for investigating the protein's expression, localization, and function:

• Western Blotting (WB): The most common application, used to detect RHBDL2 protein expression levels in cell or tissue lysates. This method is particularly valuable for monitoring changes in RHBDL2 expression under different experimental conditions, such as in suspension cultures that mimic anoikis .

• Immunohistochemistry (IHC): Used to visualize the distribution and localization of RHBDL2 in tissue sections, helping researchers understand its expression patterns in different cellular contexts .

• FLISA (Fluorescent Linked Immunosorbent Assay): A specialized application for quantitative measurement of RHBDL2 in complex biological samples .

• Immunofluorescence: Used for subcellular localization studies to confirm RHBDL2's presence at the plasma membrane and potentially other cellular compartments.

• Co-immunoprecipitation: Applied to investigate protein-protein interactions between RHBDL2 and its potential substrates or regulatory partners.

What criteria should be considered when selecting an appropriate RHBDL2 antibody for specific experimental applications?

When selecting an RHBDL2 antibody, researchers should consider:

• Antibody Specificity: Determine whether the antibody recognizes specific regions of RHBDL2 (e.g., N-terminal region) that might be important for your research question .

• Species Reactivity: Verify the antibody's reactivity with your experimental model organism. Available antibodies may recognize human, mouse, or rat RHBDL2, with some offering cross-reactivity across multiple species .

• Validated Applications: Confirm that the antibody has been validated for your specific application (WB, IHC, etc.) .

• Conjugation/Tag: Consider whether an unconjugated antibody or one with a specific tag (e.g., PE) is more appropriate for your experimental design .

• Citation Record: Antibodies with published citations generally offer more reliability and reproducibility for specific applications .

• Monoclonal vs. Polyclonal: Monoclonal antibodies offer higher specificity but potentially lower sensitivity compared to polyclonal antibodies.

How can RHBDL2 antibodies be utilized to investigate cancer metastasis mechanisms?

RHBDL2 antibodies are invaluable tools for studying cancer metastasis through several methodological approaches:

• Monitoring RHBDL2 Expression in Metastatic Models: Western blotting with RHBDL2 antibodies can be used to track changes in expression levels during suspension cultures that mimic the homeless environment encountered by metastatic cells. Research has shown that RHBDL2 protein levels increase in a time-dependent manner in highly invasive breast cancer cells (MDA-MB-231) and suspension-adapted cervical cancer cells (HeLa S3), but not in less aggressive counterparts .

• Correlation with Anoikis Resistance: RHBDL2 antibodies can be used to establish correlations between RHBDL2 expression and anoikis resistance markers such as cleaved caspase-3. This approach has revealed that RHBDL2 overexpression is associated with reduced cleaved caspase-3 levels and fewer apoptotic cells in suspension cultures .

• Therapeutic Target Validation: In knockdown studies, RHBDL2 antibodies can confirm successful protein depletion before assessing effects on anoikis sensitivity. Research has demonstrated that RHBDL2 inhibition via shRNA increases cleaved caspase-3 and enhances apoptosis in suspension cultures, highlighting RHBDL2 as a potential therapeutic target .

• Tissue Microarray Analysis: RHBDL2 antibodies can be applied to tumor tissue microarrays to correlate expression levels with metastatic potential, patient outcomes, and other clinicopathological parameters.

What methodological considerations should be addressed when designing experiments to study RHBDL2-mediated proteolysis?

When investigating RHBDL2-mediated proteolysis, researchers should consider:

• Appropriate Controls: Include both wild-type RHBDL2 and catalytically inactive mutant (serine to alanine) to distinguish between proteolytic and non-proteolytic effects. This approach revealed that substrates like EGF, BCAM, Spint-1, DDR1, CLCP1, Cadm1, and KIRREL are specifically cleaved by active RHBDL2 .

• Detection of Both Substrate and Products: Design experiments to track both the membrane-bound substrate in cell lysates and the released ectodomain in conditioned media to confirm proteolytic cleavage. This dual detection method provides more robust evidence of RHBDL2 activity .

• Quantitative Proteomics Approaches: Consider utilizing SILAC (Stable Isotope Labeling with Amino acids in Cell culture) to objectively identify substrates, as demonstrated in studies that revealed novel RHBDL2 substrates like IL6R, Spint-1, and DDR1 .

• Validation of Endogenous Activity: After identifying potential substrates through overexpression studies, validate whether endogenously expressed RHBDL2 can cleave these substrates under physiological conditions .

• Substrate Specificity Assessment: Design comparative experiments with other proteases (e.g., ADAM metalloproteases) to determine substrate specificity and potential functional redundancy or uniqueness .

How can researchers differentiate between RHBDL2 and metalloprotease-mediated substrate cleavage in experimental settings?

Differentiating between RHBDL2 and metalloprotease-mediated cleavage requires several experimental approaches:

• Selective Inhibition Studies: Utilize specific inhibitors for different protease classes:

  • DCI (dichloroisocoumarin) for inhibiting rhomboid proteases like RHBDL2

  • GM6001, TIMP3, or other metalloprotease inhibitors for ADAMs

  • Compare substrate cleavage patterns under different inhibitor conditions

• Gene Silencing Approaches: Use targeted shRNA or CRISPR/Cas9 to specifically knockdown RHBDL2 or specific metalloproteases, then assess substrate cleavage patterns .

• Cleavage Site Analysis: Identify the precise cleavage sites in substrates using mass spectrometry or N-terminal sequencing of shed products, as metalloproteases and rhomboid proteases often cleave at different positions within the substrate.

• Substrate Mutant Construction: Generate substrate mutants that are resistant to specific proteases by mutating their cleavage sites, then assess whether they remain susceptible to other proteases.

• Double Knockdown/Inhibition Experiments: Simultaneously inhibit both RHBDL2 and metalloproteases to identify potentially redundant or synergistic effects on substrate cleavage.

What cell culture models are most appropriate for studying RHBDL2 function in anoikis resistance?

The following cell culture models have been validated for studying RHBDL2's role in anoikis resistance:

• Suspension Culture Systems:

  • Poly-HEMA coated plates to prevent cell attachment

  • Hanging drop cultures

  • Ultra-low attachment plates

  These systems force cells into suspension, mimicking the homeless environment encountered by metastatic cells. Studies have shown that highly invasive breast cancer cells (MDA-MB-231) and suspension-adapted cervical cancer cells (HeLa S3) upregulate RHBDL2 under these conditions .

• Cell Line Selection:

  • Aggressive/Metastatic Cell Lines: MDA-MB-231 (highly invasive breast cancer) and HeLa S3 (suspension-adapted cervical cancer) show increased RHBDL2 expression and anoikis resistance during suspension culture .

  • Less Aggressive Counterparts: MCF-7 (less invasive breast cancer) and HeLa (non-adapted cervical cancer) show limited RHBDL2 upregulation and higher apoptosis rates during suspension culture .

  • Non-transformed Epithelial Cells: Primary keratinocytes or immortalized non-tumorigenic epithelial cell lines can be transfected with RHBDL2 to study its effects on anoikis sensitivity .

• Time-Course Experiments: Optimal results are observed when analyzing RHBDL2 expression and anoikis markers over 48-hour suspension cultures, as time-dependent changes have been documented .

How can quantitative proteomics approaches be optimized for RHBDL2 substrate identification?

To optimize quantitative proteomics for RHBDL2 substrate identification:

• SILAC-Based Approach: Use Stable Isotope Labeling with Amino acids in Cell culture to differentially label control and RHBDL2-expressing cells. This approach has successfully identified novel RHBDL2 substrates by comparing secretome profiles .

• Experimental Design Considerations:

  • Express wild-type RHBDL2 and catalytically inactive mutant (S/A) as controls

  • Use cell lines with low endogenous RHBDL2 expression (e.g., HEK293ET cells)

  • Collect both conditioned media (secretome) and cell lysates

• Validation Strategy:

  • Confirm quantitative proteomics hits with orthogonal methods (e.g., Western blotting)

  • Establish correlation between SILAC ratios and observable cleavage intensity

  • Include known substrates (e.g., EGF) as positive controls

• Data Analysis Parameters:

  • Focus on transmembrane proteins enriched in the secretome of RHBDL2-expressing cells

  • Consider the type I transmembrane topology as a common feature of RHBDL2 substrates

  • Apply appropriate statistical thresholds to identify significant changes

What approaches are recommended for investigating contradictory data regarding RHBDL2 function?

When facing contradictory data about RHBDL2 function, consider these methodological approaches:

• Cell Type-Specific Effects: Examine RHBDL2 function across multiple cell types, as its activity may vary based on cellular context. For example, RHBDL2 expression patterns differ significantly between aggressive and non-aggressive cancer cell lines during suspension culture .

• Substrate Availability Assessment: Determine whether contradictory findings might result from differential expression of RHBDL2 substrates across experimental systems. The presence or absence of specific substrates can significantly impact observable RHBDL2 functions .

• Compensatory Mechanism Evaluation: Investigate whether other proteases (e.g., ADAMs) might compensate for RHBDL2 in certain contexts, potentially masking its effects. Some RHBDL2 substrates can also be shed by metalloproteases, suggesting potential redundancy .

• Technical Variation Analysis:

  • Antibody specificity issues: Different antibodies might recognize different epitopes

  • Protein tags: N- or C-terminal tags might affect RHBDL2 activity or localization

  • Expression levels: Overexpression artifacts versus physiological expression

• Integrated Multi-Omics Approach: Combine proteomics with transcriptomics and functional assays to build a more comprehensive understanding of RHBDL2 biology across experimental conditions.

What is the current understanding of RHBDL2 substrate specificity compared to other proteases?

RHBDL2 demonstrates distinct substrate specificity compared to other proteases:

• RHBDL2-Specific Substrates: Quantitative proteomics has identified several substrates specifically cleaved by RHBDL2 but not other rhomboid proteases or metalloproteases, including:

  • Interleukin-6 receptor (IL6R)

  • Cell surface protease inhibitor Spint-1 (HAI-1)

  • Collagen receptor tyrosine kinase DDR1

  • CLCP1/DCBLD2

  • KIRREL (Neph1)

  • BCAM

• Substrates with Overlapping Specificity: Some RHBDL2 substrates can also be cleaved by ADAM metalloproteases, suggesting functional redundancy in certain pathways:

  • Thrombomodulin

  • B-type ephrins

  • Epidermal growth factor (EGF)

• Structural Determinants: RHBDL2 appears to preferentially target type I membrane proteins (extracellular N-terminus, single transmembrane domain, cytoplasmic C-terminus), although the specific sequence or structural motifs that determine cleavage susceptibility remain to be fully elucidated .

• Tissue-Specific Expression: RHBDL2 expression is restricted to specific epithelial tissues including intestine, stomach, prostate, and skin, suggesting tissue-specific functions distinct from more broadly expressed proteases .

How can researchers design experiments to elucidate the physiological roles of RHBDL2-mediated proteolysis?

To investigate the physiological roles of RHBDL2-mediated proteolysis:

• Tissue-Specific Expression Analysis:

  • Perform comprehensive tissue expression profiling of RHBDL2 using antibodies in immunohistochemistry

  • Focus on epithelial tissues (skin, airways, digestive system) where RHBDL2 expression has been detected

  • Correlate RHBDL2 expression with substrate availability in specific tissues

• In vivo Models:

  • Generate tissue-specific RHBDL2 knockout or knockin mouse models

  • Analyze phenotypes related to epithelial homeostasis, particularly in tissues with high RHBDL2 expression

  • Examine the impact on known substrate processing in vivo

• Signaling Pathway Analysis:

  • Investigate how RHBDL2-mediated cleavage affects downstream signaling of substrates like IL6R, DDR1, and EGFR

  • Use specific pathway inhibitors (e.g., EGFR inhibitors) in combination with RHBDL2 modulation to dissect signaling mechanisms

  • Employ phospho-specific antibodies to monitor activation of downstream effectors like FAK (focal adhesion kinase)

• Disease Model Studies:

  • Analyze RHBDL2 expression in cancer models, particularly those involving anoikis resistance

  • Investigate RHBDL2 function in wound healing and epithelial regeneration

  • Explore potential roles in inflammatory conditions related to IL6R shedding

What methodological approaches can be used to identify novel functions of RHBDL2 beyond its established role in anoikis resistance?

To discover novel RHBDL2 functions:

• Unbiased Interactome Analysis:

  • Perform immunoprecipitation of RHBDL2 followed by mass spectrometry

  • Use proximity labeling techniques (BioID, APEX) to identify proteins in close proximity to RHBDL2

  • Map the temporal dynamics of the RHBDL2 interactome under different cellular conditions

• Subcellular Localization Studies:

  • Use super-resolution microscopy to precisely map RHBDL2 localization

  • Investigate potential translocation of RHBDL2 under different cellular stresses

  • Determine whether RHBDL2 localizes to specific membrane microdomains or organelles beyond the plasma membrane

• Functional Genomics Screens:

  • Perform CRISPR/Cas9 screens to identify synthetic lethal interactions with RHBDL2

  • Use transcriptome profiling to identify gene expression changes upon RHBDL2 modulation

  • Conduct phenotypic screens under various stress conditions in RHBDL2-modified cells

• Non-Proteolytic Function Investigation:

  • Generate catalytically inactive RHBDL2 mutants to distinguish between proteolytic and potential non-proteolytic functions

  • Investigate whether RHBDL2 can act as a scaffold for signaling complexes

  • Explore potential regulatory roles in membrane protein trafficking

What are the most effective validation methods to confirm RHBDL2 antibody specificity?

To validate RHBDL2 antibody specificity:

• Genetic Validation:

  • Use RHBDL2 knockout cells (CRISPR/Cas9-generated) as negative controls

  • Employ RHBDL2 knockdown cells (shRNA or siRNA) to verify signal reduction

  • Test in cells with confirmed RHBDL2 overexpression as positive controls

• Multi-Antibody Approach:

  • Compare signals from multiple antibodies targeting different RHBDL2 epitopes

  • Include antibodies against N-terminal and other regions of RHBDL2

  • Verify consistent detection patterns across different antibodies

• Immunoprecipitation-Mass Spectrometry:

  • Perform immunoprecipitation with the RHBDL2 antibody

  • Confirm the identity of the precipitated protein by mass spectrometry

  • Verify the expected molecular weight (approximately 34 kDa)

• Cross-Reactivity Assessment:

  • Test antibody against related rhomboid family proteins

  • Verify species specificity when working with human, mouse, or rat samples

  • Perform peptide competition assays with the immunizing peptide

• Application-Specific Validation:

  • For Western blotting: Confirm single band at expected molecular weight

  • For IHC/IF: Include peptide blocking controls and compare with mRNA expression patterns

  • For functional studies: Verify detection of active vs. inactive RHBDL2 variants

How can researchers overcome challenges in detecting low-abundance RHBDL2 in endogenous systems?

To improve detection of endogenous RHBDL2:

• Sample Enrichment Techniques:

  • Perform subcellular fractionation to concentrate membrane proteins

  • Use immunoprecipitation to enrich RHBDL2 before detection

  • Apply plasma membrane isolation protocols to enhance signal-to-noise ratio

• Signal Amplification Methods:

  • Utilize high-sensitivity ECL substrates for Western blotting

  • Implement tyramide signal amplification for immunohistochemistry

  • Consider proximity ligation assays for detecting protein-protein interactions

• Optimized Lysis Conditions:

  • Use detergents optimized for membrane protein extraction (e.g., CHAPS, digitonin)

  • Include protease inhibitors to prevent degradation during sample preparation

  • Optimize buffer conditions to maintain RHBDL2 stability

• Cell System Selection:

  • Focus on cell types with documented RHBDL2 expression (epithelial cells)

  • Consider stress conditions that upregulate RHBDL2 (e.g., suspension culture for cancer cells)

  • Use cell models relevant to RHBDL2 function (e.g., HeLa S3 vs. HeLa)

• Increased Sample Input:

  • Load more protein for Western blotting

  • Process larger volumes of conditioned media to detect shed substrates

  • Use larger numbers of cells for immunoprecipitation experiments

What strategies can be employed to study RHBDL2 activity in complex physiological environments?

For studying RHBDL2 in complex physiological contexts:

• Ex Vivo Tissue Models:

  • Utilize organoid cultures from relevant tissues (skin, intestine, airways)

  • Employ precision-cut tissue slices to maintain tissue architecture

  • Develop 3D co-culture systems to model epithelial-stromal interactions

• Activity-Based Protein Profiling:

  • Develop activity-based probes specific for RHBDL2's serine protease activity

  • Apply in situ labeling approaches to detect active RHBDL2 in tissues

  • Compare activity profiles across different physiological and pathological states

• Substrate Reporter Systems:

  • Generate fluorescent or luminescent reporter constructs based on RHBDL2 substrates

  • Develop FRET-based reporters to monitor RHBDL2 activity in real-time

  • Apply these reporters in relevant cell types or ex vivo models

• Conditional Expression Systems:

  • Implement inducible RHBDL2 expression or knockdown in specific cell types

  • Use tissue-specific promoters to drive expression in relevant physiological contexts

  • Employ temporal control to study RHBDL2 function during specific developmental or disease stages

• Multiplex Imaging Approaches:

  • Apply multiplexed immunofluorescence to simultaneously visualize RHBDL2, its substrates, and relevant signaling components

  • Use imaging mass cytometry for high-dimensional analysis of RHBDL2 in tissue contexts

  • Implement spatial transcriptomics to correlate RHBDL2 protein expression with local gene expression profiles

How might RHBDL2 antibodies be utilized in developing potential therapeutic approaches for cancer?

RHBDL2 antibodies offer several potential therapeutic applications in cancer research:

• Target Validation Studies:

  • Use RHBDL2 antibodies to confirm expression in patient-derived xenografts or tumor samples

  • Correlate RHBDL2 expression with clinical outcomes and metastatic potential

  • Identify cancer types where RHBDL2-targeting therapies might be most effective

• Therapeutic Antibody Development:

  • Generate function-blocking antibodies targeting RHBDL2's extracellular domains

  • Develop antibody-drug conjugates to deliver cytotoxic agents to RHBDL2-expressing cells

  • Create bispecific antibodies linking RHBDL2 to immune effector cells

• Companion Diagnostic Development:

  • Use validated RHBDL2 antibodies to create immunohistochemistry-based diagnostics

  • Identify patients likely to benefit from RHBDL2-targeting therapies

  • Monitor treatment efficacy through changes in RHBDL2 expression or activity

• Mechanism of Action Studies:

  • Investigate how RHBDL2 inhibition affects anoikis sensitivity in metastatic models

  • Examine consequences of blocking RHBDL2-mediated EGFR activation

  • Assess combination approaches with existing therapies targeting EGFR or downstream pathways

• Biomarker Research:

  • Evaluate RHBDL2 and its cleaved substrates as potential biomarkers for metastatic potential

  • Develop liquid biopsy approaches to detect shed RHBDL2 substrates

  • Correlate changes in RHBDL2 substrate profiles with disease progression or treatment response

What experimental approaches are recommended for investigating RHBDL2's role in epithelial homeostasis?

To elucidate RHBDL2's role in epithelial homeostasis:

• Epithelial Wound Healing Models:

  • Utilize scratch assays with RHBDL2 modulation in epithelial monolayers

  • Apply live cell imaging to track cell migration and proliferation

  • Assess RHBDL2's impact on wound closure kinetics

• Barrier Function Assessment:

  • Measure transepithelial electrical resistance in RHBDL2-modulated epithelial models

  • Use permeability assays to assess barrier integrity

  • Investigate effects on tight junction and adherens junction proteins

• Differentiation Models:

  • Study RHBDL2's role during air-liquid interface culture of airway epithelial cells

  • Examine effects on intestinal organoid formation and differentiation

  • Assess impact on epidermal differentiation in 3D skin models

• Cell-Cell Communication Analysis:

  • Investigate how RHBDL2-mediated shedding affects juxtacrine signaling

  • Examine effects on N-Cadherin processing and cell-cell adhesion

  • Study consequences for epithelial-mesenchymal transition processes

• Response to Stress:

  • Challenge epithelial models with relevant stressors (mechanical, chemical, inflammatory)

  • Assess RHBDL2's role in adaptation and recovery

  • Investigate potential protective functions against environmental challenges