TRPC4AP Antibody

What is TRPC4AP and what are its key biological functions?

TRPC4AP (Transient Receptor Potential Cation Channel, Subfamily C, Member 4 Associated Protein) is a 797 amino acid protein that functions as an adaptor protein interacting with the TRPC4 ion channel . It serves as a substrate-recognition component of a DCX (DDB1-CUL4-X-box) E3 ubiquitin-protein ligase complex required for cell cycle control . TRPC4AP specifically mediates the polyubiquitination and subsequent degradation of MYC through the DesCEND (destruction via C-end degrons) pathway .

Additionally, TRPC4AP:

• Participates in the activation of NFKB1 in response to TNFRSF1A ligation

• Links TNFRSF1A to the IKK signalosome

• Is involved in JNK activation via interaction with TRAF2

• Contributes to endoplasmic reticulum Ca²⁺ storage reduction

The protein is widely expressed across tissues, with highest expression reported in liver, heart, testis, and brain .

How should researchers select an appropriate TRPC4AP antibody for their experiments?

When selecting a TRPC4AP antibody, researchers should consider:

Epitope targeting: Different antibodies target distinct regions of TRPC4AP:

• N-terminal region antibodies (aa 1-50)

• Middle region antibodies (aa 50-300)

• C-terminal region antibodies

Host species and format:

• Rabbit polyclonal antibodies provide high sensitivity but may have batch-to-batch variation

• Mouse monoclonal antibodies offer high specificity and reproducibility

• Goat antibodies are available for specialized applications

Validated applications:

Species reactivity: Verify cross-reactivity with your experimental model (human, mouse, rat, etc.) .

What are the optimal conditions for detecting TRPC4AP by Western blotting?

To optimize Western blot detection of TRPC4AP:

Sample preparation:

• Use tissues with known high expression (heart, liver, brain) as positive controls

• For cell lines, HepG2 and HuH-7 cells show reliable TRPC4AP expression

• Lyse cells in RIPA buffer with protease inhibitors

Electrophoresis and transfer parameters:

• Use 8-10% SDS-PAGE gels for optimal resolution of TRPC4AP (85-90 kDa)

• Transfer to PVDF membranes at 100V for 90 minutes in 10% methanol transfer buffer

Antibody incubation:

• Block membranes with 5% non-fat milk in TBST for 1 hour

• Incubate with primary antibody at appropriate dilution (1:200-1:3000) overnight at 4°C

• Use appropriate secondary antibody (typically 1:5000-1:10000) for 1 hour at room temperature

Detection considerations:

• Expected molecular weight is 85-90 kDa

• Multiple bands may indicate isoforms or post-translational modifications

• For enhanced specificity, consider antibody combinations targeting different epitopes

What are the recommended protocols for immunohistochemical detection of TRPC4AP?

For effective immunohistochemical detection of TRPC4AP:

Tissue preparation and fixation:

• 10% neutral buffered formalin fixation for 24-48 hours

• Paraffin embedding and sectioning at 4-5 μm thickness

Antigen retrieval methods:

• TE buffer (pH 9.0) is the preferred method

• Alternative: citrate buffer (pH 6.0)

• Heat-induced epitope retrieval: 95-98°C for 15-20 minutes

Staining protocol:

• Block endogenous peroxidase with 3% H₂O₂ in methanol

• Block non-specific binding with normal serum

• Primary antibody dilution range: 1:50-1:500

• Incubate at 4°C overnight or at room temperature for 1-2 hours

• Detection using appropriate secondary antibody and visualization system

Positive control tissues:

• Human breast cancer tissue

• Human ovary cancer tissue

• Human colon cancer tissue

• Human liver tissue

How can researchers investigate the interaction between TRPC4 and TRPC4AP?

To study the TRPC4-TRPC4AP interaction:

Co-immunoprecipitation approaches:

• Use anti-TRPC4 antibody to pull down the complex, then probe with anti-TRPC4AP

• Alternatively, immunoprecipitate with anti-TRPC4AP and detect TRPC4

• Cross-validate results by performing reciprocal co-IPs

Proximity ligation assay (PLA):

• Enables visualization of protein interactions in situ with subcellular resolution

• Use combinations of antibodies from different host species (mouse anti-TRPC4 and rabbit anti-TRPC4AP)

Molecular modeling:

• Recent research has employed AlphaFold 3 to model the TRPC4-TRPC4AP complex

• Analysis of interfacial interactions identified crucial interactions between arginine at position 730 on TRPC4 and glutamic acid on TRPC4AP

CRISPR-based approaches:

• Generate knockout or mutant cell lines targeting specific domains

• The first and second exons of both genes have been targeted successfully

What is the role of TRPC4AP in the hydrogen-induced calcium influx pathway?

Recent research has revealed that TRPC4AP is involved in hydrogen-induced calcium influx:

Molecular mechanism:

• H₂ modulates TRPC4 channel activity by targeting the CIRB domain within the TRPC4-TRPC4AP complex

• Arginine residues at positions 730 (Arg730) and 731 (Arg731) of TRPC4 play a pivotal role in H₂-mediated Ca²⁺ transmembrane transport

• These residues form critical hydrogen-bond networks that are essential for channel function

Experimental approaches to study this pathway:

• Calcium imaging techniques to measure intracellular calcium levels

• Site-directed mutagenesis of Arg730 and Arg731 to evaluate their functional significance

• Structural analysis using computational modeling and molecular dynamics simulations

Potential therapeutic relevance:

• TRPC4AP has been identified as a unique molecular target for hydrogen therapy

• This pathway may be relevant to developing targeted therapies for degenerative diseases

Why might researchers observe multiple bands or inconsistent results when using TRPC4AP antibodies?

Multiple bands or inconsistent results may occur due to:

Isoform detection:

• TRPC4AP exists as multiple alternatively spliced isoforms

• Different antibodies may recognize distinct isoforms

Post-translational modifications:

• Phosphorylation, ubiquitination, or other modifications may alter migration patterns

• These modifications can change in response to cellular signaling

Technical factors:

• Sample preparation variations (different lysis buffers)

• Protein degradation during extraction

• Non-specific binding of antibodies

Troubleshooting approaches:

• Verify antibody specificity using knockout/knockdown controls

• Test multiple antibodies targeting different epitopes

• Optimize blocking conditions (5% milk vs. 3-5% BSA)

• Adjust antibody concentrations based on signal-to-noise ratio

• Include protease and phosphatase inhibitors in lysis buffers

What methods can researchers use to validate TRPC4AP antibody specificity?

To validate TRPC4AP antibody specificity:

Genetic approaches:

• siRNA or shRNA knockdown of TRPC4AP

• CRISPR/Cas9-mediated knockout cell lines

• Overexpression systems with tagged TRPC4AP constructs

Biochemical validation:

• Pre-absorption with immunizing peptide

• Comparison of multiple antibodies targeting different epitopes

• Mass spectrometry confirmation of immunoprecipitated proteins

Western blot considerations:

• Observe expected molecular weight (85-90 kDa)

• Compare staining patterns across multiple tissue/cell types with known expression levels

• Include positive controls (heart tissue, HepG2 cells)

Documentation requirements:

• Record complete antibody information (catalog number, lot, dilution)

• Maintain validation records for reproducibility

• Include appropriate controls in publications

How might researchers investigate the role of TRPC4AP in the DesCEND pathway for targeted protein degradation?

To explore TRPC4AP's role in the DesCEND pathway:

Protein interaction studies:

• Identify proteins with the R-3 motif (arginine at position -3 from C-terminus) that may be TRPC4AP targets

• Perform Co-IP experiments to validate potential interactions

• Use proximity labeling approaches (BioID, APEX) to identify novel interaction partners

Functional assays:

• Measure ubiquitination levels of target proteins (e.g., MYC) in TRPC4AP-depleted cells

• Employ proteasome inhibitors to stabilize ubiquitinated intermediates

• Conduct pulse-chase experiments to measure protein half-life changes

Structural approaches:

• Determine the structural basis for TRPC4AP recognition of the R-3 motif

• Employ mutagenesis to identify critical residues in the substrate recognition domain

• Use structural biology techniques to characterize the DCX(TRPC4AP) complex

Therapeutic relevance:

• Evaluate the potential of targeting TRPC4AP for modulating MYC levels in cancer models

• Develop small molecule inhibitors or peptide mimetics that disrupt specific TRPC4AP interactions

What techniques can be employed to study TRPC4AP's involvement in NFκB signaling pathways?

To investigate TRPC4AP's role in NFκB signaling:

Signaling pathway analysis:

• Monitor NFκB translocation using immunofluorescence or cellular fractionation

• Measure NFκB target gene expression via qRT-PCR or reporter assays

• Assess IκB degradation kinetics in response to TNF-α stimulation

Protein complex assembly studies:

• Characterize the interaction between TRPC4AP and TNFRSF1A

• Investigate TRPC4AP's association with the IKK signalosome

• Use temporal analysis to determine sequential recruitment of signaling components

Functional manipulation:

• Generate TRPC4AP domain mutants to identify regions critical for NFκB activation

• Employ inducible knockdown/knockout systems to assess temporal requirements

• Use pharmacological inhibitors of specific pathway components to position TRPC4AP within the signaling cascade

Disease models:

• Evaluate TRPC4AP's role in inflammatory conditions

• Assess how TRPC4AP expression correlates with NFκB activity in pathological samples

• Determine if TRPC4AP modulation affects inflammatory responses in vivo