RHBDD3 Antibody, Biotin conjugated

What is RHBDD3 and what are its primary biological functions?

RHBDD3, also known as pituitary tumor apoptosis gene (PTAG), is a membrane-bound protease belonging to the rhomboid family of proteins. It functions primarily as a negative regulator of immune responses, particularly in:

• Toll-like receptor 3 (TLR3)-mediated natural killer (NK) cell activation

• Attenuation of acute inflammation responses

• Regulation of cell-cell interactions in immune contexts

RHBDD3 achieves its immunomodulatory effects by interacting with DNAX activation protein of 12 kDa (DAP12) and promoting its degradation, which inhibits MAPK signaling pathways in TLR3-triggered NK cells . Initial studies identified RHBDD3 as a pituitary tumor apoptosis gene with reduced expression in certain pituitary adenomas. Subsequent research discovered its expression is also decreased in several primary colorectal tumors, suggesting that loss of RHBDD3 contributes to a blunted apoptotic response and may predispose cells toward malignant transformation .

In TLR3-stimulated conditions, RHBDD3 is selectively upregulated in NK cells, acting as a feedback inhibitor that prevents excessive immune activation .

What applications are RHBDD3 Biotin-conjugated antibodies suitable for?

RHBDD3 Biotin-conjugated antibodies are designed for multiple research applications, with varying protocols and optimization requirements:

The biotin conjugation leverages the high-affinity biotin-avidin interaction (Kd ~10⁻¹⁵ M), allowing precise and sensitive detection when coupled with streptavidin-conjugated detection systems . This approach enhances signal strength while maintaining background control when properly optimized.

How should researchers validate RHBDD3 Biotin-conjugated antibody specificity?

Validating antibody specificity is crucial for generating reliable research data. For RHBDD3 Biotin-conjugated antibodies, a multi-step validation approach is recommended:

• Positive and negative control tissues/cells:

  • Positive controls: Human NK cells after poly(I:C) stimulation (which upregulates RHBDD3)

  • Negative controls: Tissues from RHBDD3-deficient (Rhbdd3⁻/⁻) mice

• Western blot validation:

  • Verify a single band at the expected molecular weight (~43 kDa)

  • Perform peptide competition assays using the immunizing peptide

  • Compare signal pattern with documented results showing RHBDD3 in rat lung tissue lysate

• Epitope mapping confirmation:

  • Confirm recognition of the target epitope (AA 304-323 or AA 101-200, depending on antibody)

  • Test cross-reactivity with other rhomboid family proteins using recombinant proteins

• RNA interference:

  • Perform siRNA knockdown of RHBDD3 and confirm reduced antibody signal

  • Include non-targeting siRNA controls

This comprehensive validation ensures that experimental observations genuinely reflect RHBDD3 biology rather than non-specific binding or cross-reactivity with related proteins like RHBDD1, RHBDD2, or RHBDL proteins .

What is the optimal experimental design for studying RHBDD3's role in NK cell regulation using biotin-conjugated antibodies?

When investigating RHBDD3's role in NK cell regulation, consider this experimental framework:

• Cell isolation and culture:

  • Isolate NK cells from human or mouse splenocytes

  • Culture in appropriate media supplemented with IL-2

• TLR3 stimulation protocol:

  • Treat NK cells with poly(I:C) (10-50 μg/ml) for 6-24 hours

  • Include IL-12/15 stimulation as comparative control (which doesn't affect RHBDD3 expression)

• Co-culture experiments:

  • Set up NK cell-DC (dendritic cell) co-cultures

  • Implement Transwell systems to distinguish contact-dependent from soluble factor-mediated effects

• Detection and analysis:

  • Use RHBDD3 Biotin-conjugated antibody (1:200-1:500) for immunofluorescence

  • Perform co-immunoprecipitation to study RHBDD3-DAP12 interactions

  • Measure NK cell activation markers (IFN-γ, granzyme B) by flow cytometry

• Functional assessment:

  • Cytotoxicity assays against appropriate target cells

  • Cytokine measurement in culture supernatants

This experimental design allows for comprehensive analysis of how RHBDD3 negatively regulates TLR3-mediated NK cell activation, particularly through its interaction with DAP12 and subsequent effects on MAPK signaling pathways .

How does RHBDD3 interact with DAP12, and how can this interaction be investigated?

RHBDD3 negatively regulates NK cell activation by interacting with DAP12 (DNAX activation protein of 12 kDa) and promoting its degradation. This interaction can be investigated through:

• Co-immunoprecipitation studies:

  • Immunoprecipitate with anti-RHBDD3 antibody and probe for DAP12

  • Reverse IP with anti-DAP12 antibody and probe for RHBDD3

  • Western blot analysis shows that RHBDD3 interacts with DAP12 in poly(I:C)-activated NK cells

• Confocal microscopy:

  • Use RHBDD3 Biotin-conjugated antibody with streptavidin-fluorophore

  • Co-stain with fluorescently labeled anti-DAP12 antibody

  • Analyze colocalization patterns before and after TLR3 stimulation

  • Research has shown that DAP12 and RHBDD3 increase and aggregate significantly after poly(I:C) stimulation

• Degradation analysis:

  • Compare DAP12 protein levels in Rhbdd3⁺/⁺ vs. Rhbdd3⁻/⁻ cells

  • Use proteasome inhibitors (e.g., MG132) to determine degradation mechanism

  • Measure DAP12 mRNA levels to confirm post-transcriptional regulation

  • Studies show that DAP12 protein (but not mRNA) levels are significantly elevated after Rhbdd3 knockdown or deletion

• Functional consequences:

  • Analyze MAPK activation downstream of DAP12

  • Measure NK cell activation markers in the presence/absence of functional RHBDD3

These methods comprehensively assess the RHBDD3-DAP12 interaction and its functional significance in regulating NK cell activation through the MAPK signaling pathway .

What considerations are important when using RHBDD3 Biotin-conjugated antibodies in immunohistochemistry?

When performing immunohistochemistry (IHC) with RHBDD3 Biotin-conjugated antibodies, researchers should address these critical considerations:

• Tissue preparation:

  • For IHC-P: Use 10% neutral-buffered formalin fixation (12-24 hours)

  • For IHC-F: Flash-freeze tissues in OCT compound

  • Section thickness: 5-7 μm optimal for signal resolution

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

  • Pressure cooker method (125°C for 3 minutes) provides consistent results

  • Allow 20-30 minutes cooling before proceeding

• Endogenous biotin blocking:

  • Critical step: Block endogenous biotin using avidin/biotin blocking kit

  • Apply avidin solution (15 minutes) followed by biotin solution (15 minutes)

  • Omitting this step will result in high background in biotin-rich tissues (liver, kidney)

• Antibody dilution:

  • For paraffin sections (IHC-P): 1:200-1:400 dilution range

  • For frozen sections (IHC-F): 1:100-1:500 dilution range

  • Titration experiments advised for each new tissue type

• Detection system:

  • Streptavidin-HRP or streptavidin-AP systems recommended

  • DAB or AEC chromogens for brightfield microscopy

  • Counterstain nuclei with hematoxylin (2-3 minutes exposure)

• Controls:

  • Positive control: Rat lung tissue (verified RHBDD3 expression)

  • Negative control: Omit primary antibody

  • Peptide competition control: Pre-incubate antibody with immunizing peptide

Following these guidelines enables accurate localization of RHBDD3 protein while minimizing background and non-specific staining artifacts.

How can RHBDD3 Biotin-conjugated antibodies be utilized to investigate RHBDD3's role in acute liver inflammation?

RHBDD3 plays a critical role in attenuating TLR3-triggered acute liver inflammation. A comprehensive experimental design to investigate this function using RHBDD3 Biotin-conjugated antibodies would include:

• Animal model implementation:

  • Establish acute liver inflammation using poly(I:C)/D-GalN injection

  • Compare responses in wild-type vs. Rhbdd3⁻/⁻ mice

  • Design intervention groups testing NK cell depletion using anti-NK1.1 antibodies

• Tissue analysis workflow:

  • Collect liver tissue at multiple timepoints (6, 12, 24 hours)

  • Section for IHC and prepare protein/RNA extracts

  • Process serum for liver enzymes (ALT, AST) and cytokine analysis

• RHBDD3 detection protocol:

  • Use RHBDD3 Biotin-conjugated antibody (1:200) for IHC

  • Double-stain with NK cell markers (NK1.1) and Kupffer cell markers (F4/80)

  • Analyze cell-specific expression and localization patterns

• Functional analysis:

  • Measure inflammatory infiltrates and tissue damage scores

  • Quantify hepatic IL-6 and other inflammatory cytokines

  • Assess NK cell activation status and accumulation in liver

• Mechanistic investigation:

  • Perform adoptive transfer experiments with Rhbdd3⁺/⁺ or Rhbdd3⁻/⁻ NK cells

  • Use clodronate liposomes to deplete Kupffer cells and assess effect on inflammation

  • Analyze NK cell-Kupffer cell interactions using confocal microscopy

Research has shown that Rhbdd3⁻/⁻ mice exhibit exaggerated liver damage with elevated ALT/AST, increased inflammatory infiltrates, higher hepatic IL-6, and accelerated mortality following poly(I:C) challenge . RHBDD3 Biotin-conjugated antibodies allow precise localization of RHBDD3 protein in this disease model.

What are the recommended approaches for analyzing RHBDD3 expression in NK cells following TLR3 stimulation?

Analysis of RHBDD3 expression in NK cells after TLR3 stimulation requires specific methodological approaches:

• Cell stimulation protocol:

  • Isolate NK cells (magnetic separation or FACS sorting)

  • Stimulate with poly(I:C) (10-50 μg/ml) for defined timepoints (1-24h)

  • Include IL-12/15 stimulation as control (doesn't affect RHBDD3 expression)

  • Include unstimulated controls at each timepoint

• Protein expression analysis:

  • Western blot using 30-50 μg total protein per lane

  • Use RHBDD3 Biotin-conjugated antibody (1:500-1:1000)

  • Streptavidin-HRP detection system with ECL substrate

  • Normalize to β-actin or GAPDH housekeeping proteins

• RNA expression analysis:

  • qRT-PCR for RHBDD3 mRNA quantification

  • Design primers spanning exon-exon junctions

  • Normalize to stable reference genes (GAPDH, ACTB, HPRT)

• Flow cytometry approach:

  • Fix and permeabilize NK cells (paraformaldehyde/saponin)

  • Stain with RHBDD3 Biotin-conjugated antibody

  • Detect with streptavidin-fluorophore conjugate

  • Co-stain with NK markers (CD56, NK1.1) and activation markers

• Single-cell analysis:

  • Immunofluorescence microscopy of adhered NK cells

  • Confocal imaging to determine subcellular localization

  • Co-staining with DAP12 to assess interaction dynamics

Research has demonstrated that RHBDD3 expression increases rapidly and significantly in NK cells following poly(I:C) stimulation both in vitro and in vivo, suggesting a feedback regulatory mechanism .

How can researchers design experiments to investigate RHBDD3's potential role in cancer using biotin-conjugated antibodies?

RHBDD3 was initially identified as a pituitary tumor apoptosis gene (PTAG) with reduced expression in certain tumors. To investigate its cancer-related functions:

• Expression profiling in cancer tissues:

  • Design tissue microarrays (TMAs) of various cancer types

  • Perform IHC using RHBDD3 Biotin-conjugated antibody (1:200-1:400)

  • Score expression patterns (0-3+) and correlate with clinical parameters

  • Compare with normal adjacent tissue (NAT) controls

• Functional studies in cancer cell lines:

  • Select cell lines with variable RHBDD3 expression

  • Create RHBDD3 overexpression and knockdown models

  • Analyze effects on:

    • Apoptosis (annexin V/PI staining)

    • Proliferation (MTT/BrdU assays)

    • Migration/invasion (transwell assays)

• Mechanistic investigation:

  • Assess response to apoptosis-inducing agents (e.g., bromocriptine)

  • Measure caspase activation in RHBDD3-modulated cells

  • Determine effects on DAP12 and MAPK pathway components

• In vivo tumor models:

  • Xenograft studies with RHBDD3-modulated cancer cells

  • Compare tumor growth and metastatic potential

  • Analyze tumor-infiltrating lymphocytes (TILs)

• Protein interaction studies:

  • Immunoprecipitation with RHBDD3 Biotin-conjugated antibody

  • Mass spectrometry to identify novel binding partners

  • Validation of interactions by co-IP and proximity ligation assay

Research has shown that RHBDD3 expression is reduced in certain pituitary adenomas and colorectal tumors. Overexpression of RHBDD3 in AtT20 cells increased apoptotic activity and caspase activation in response to bromocriptine, suggesting that reactivation of RHBDD3 in tumors may have therapeutic potential .

What are the key species reactivity considerations when working with RHBDD3 Biotin-conjugated antibodies?

Species reactivity is critical when selecting RHBDD3 antibodies. Different commercially available RHBDD3 Biotin-conjugated antibodies have specific reactivity profiles:

When working across species:

• Epitope conservation analysis:

  • Human and mouse RHBDD3 share approximately 87% amino acid identity

  • The AA 101-200 region shows higher conservation across species than AA 304-323

  • Antibodies targeting the more conserved regions typically have broader cross-reactivity

• Validation requirements for cross-species applications:

  • Always perform preliminary validation when using antibodies in species beyond stated reactivity

  • Include positive control samples from each species being tested

  • Consider epitope sequence alignment to predict likelihood of reactivity

• Application-specific considerations:

  • Western blot typically has higher cross-species success than IHC

  • For WB: Use species-appropriate positive control lysates

  • For IHC: Titrate antibody concentration for each species separately

• Cross-reactivity with other rhomboid family proteins:

  • Check for potential cross-reactivity with other family members (RHBDD1, RHBDD2)

  • RHBDD3 shows relationships with several rhomboid family proteins with interaction scores ranging from 0.490-0.559

Researchers should select antibodies with documented reactivity for their species of interest and perform validation studies before conducting major experiments .

How can RHBDD3 Biotin-conjugated antibodies be employed to study the role of RHBDD3 in innate antiviral signaling pathways?

RHBDD3's role in TLR3-mediated responses suggests potential involvement in antiviral immunity. To investigate this:

• Viral challenge models:

  • Challenge wild-type and Rhbdd3⁻/⁻ cells/animals with:

    • RNA viruses (influenza, VSV)

    • DNA viruses (HSV, HCMV)

  • Measure viral replication kinetics and host survival

• Signaling pathway analysis:

  • Examine type I IFN production following viral infection

  • Assess activation of:

    • IRF3/7 phosphorylation

    • NF-κB pathway components

    • TBK1/IKKε signaling complex

• NK cell-viral interaction studies:

  • Isolate NK cells from wild-type and Rhbdd3⁻/⁻ mice

  • Challenge with virus or viral components

  • Monitor NK cell cytotoxicity against virus-infected targets

  • Analyze cytokine/chemokine production profiles

• Visualization of RHBDD3 during viral infection:

  • Use RHBDD3 Biotin-conjugated antibody for immunofluorescence

  • Track subcellular localization changes during infection

  • Co-stain with viral proteins and innate immune adaptors

• Protein complex analysis:

  • Immunoprecipitate with RHBDD3 Biotin-conjugated antibody

  • Identify viral and host factors in complex by mass spectrometry

  • Validate interactions through reciprocal IP and proximity ligation assays

Research indicates that rhomboid-like pseudoproteases play roles in antiviral signaling by regulating adaptors like MITA/STING . Given RHBDD3's established role in TLR3 signaling , which is crucial for detecting viral dsRNA, investigating its function in antiviral immunity represents an important research direction.

What controls and validation steps are essential when performing co-immunoprecipitation with RHBDD3 Biotin-conjugated antibodies?

Co-immunoprecipitation (co-IP) with RHBDD3 Biotin-conjugated antibodies requires rigorous controls and validation steps:

• Pre-IP validation:

  • Confirm antibody specificity by Western blot

  • Verify RHBDD3 expression in target cells/tissues

  • Optimize lysis conditions to preserve protein interactions

• Technical controls for IP procedure:

  • Input control: 5-10% of pre-IP lysate

  • IgG control: Non-specific rabbit IgG at same concentration

  • Beads-only control: Streptavidin beads without antibody

  • Peptide competition control: Pre-incubate antibody with immunizing peptide

• Lysis buffer optimization:

  • Test multiple detergent conditions:

    • Mild: 1% NP-40 or 0.5% Triton X-100

    • Moderate: 1% Triton X-100

    • Stringent: 1% SDS (denaturing)

  • Include protease/phosphatase inhibitors

  • For membrane proteins like RHBDD3, consider digitonin or CHAPS

• IP procedure validation:

  • Confirm successful IP by blotting for RHBDD3 in IP fraction

  • Verify absence of RHBDD3 in IgG control IP

  • Ensure efficient depletion from post-IP supernatant

• Interaction validation steps:

  • Perform reverse IP with antibodies against interacting partners

  • Confirm physiological relevance with stimulus-dependent interactions

  • Validate with alternative methods (PLA, FRET, BiFC)