TNFAIP1 Antibody

What is TNFAIP1 and what are its primary functions in cellular processes?

TNFAIP1 (also known as BTB/POZ domain-containing adapter for CUL3-mediated RhoA degradation protein 2) is an immediate-early response gene induced by TNFα in endothelial cells. It functions as an adaptor protein for Cullin3-based E3 ubiquitin ligases and plays crucial roles in:

• Regulation of inflammatory responses via RhoB degradation

• DNA synthesis and cell cycle regulation

• Apoptotic processes

• Cell migration control

• Neuronal protection and function

The 316-amino acid protein contains a conserved BTB/POZ domain at its N-terminus and is developmentally regulated in a tissue-specific manner, with high expression in brain and heart tissues .

What types of TNFAIP1 antibodies are available for research applications?

Currently available TNFAIP1 antibodies include:

Most commonly used applications include Western blot, immunohistochemistry (paraffin and frozen sections), immunocytochemistry, and ELISA .

What are the optimal working dilutions for TNFAIP1 antibodies in different applications?

Application-specific dilutions vary by antibody source and experimental conditions:

For immunohistochemistry applications, a concentration of 0.25-0.5 μg/mL is typically recommended for formalin-fixed and paraffin-embedded tissues, with heat-mediated antigen retrieval required for optimal results .

How should I perform immunoprecipitation assays for detecting TNFAIP1 interactions?

For studying TNFAIP1 protein interactions, follow this optimized immunoprecipitation protocol:

• Pre-treatment: Treat cells with MG132 (proteasome inhibitor) for 6 hours prior to lysis to prevent protein degradation

• Cell lysis: Add 500 μL lysis buffer to cell dishes, collect cells, and centrifuge at 13,000 rpm for 15 minutes

• Antibody incubation: Transfer supernatant to a new tube and incubate with 1 μg TNFAIP1 antibody overnight at 4°C

• Protein pull-down: Incubate with protein A/G beads for 2 hours to pull down protein complexes

• Washing and analysis: Wash immunoprecipitate 3 times with lysis buffer and analyze by SDS-PAGE

This protocol has been validated for detecting interactions between TNFAIP1 and proteins such as RhoB, SNAP25, and CSNK2B .

How can I investigate TNFAIP1's role in inflammatory responses using antibody-based techniques?

TNFAIP1 plays a significant role in inflammatory signaling through its interaction with RhoB. To study this function:

• Knockdown/overexpression: Use siRNA for TNFAIP1 knockdown or transfect with TNFAIP1 expression plasmids

• Inflammatory stimulation: Treat cells with TNFα (10-50 ng/mL) for different time periods (0-24h)

• Protein analysis: Perform Western blot using anti-TNFAIP1 antibody (1:500-1:1000 dilution) to monitor expression levels

• Interaction studies: Use co-immunoprecipitation with TNFAIP1 antibody followed by RhoB detection

• Inflammatory markers: Monitor IL-6, IL-8 expression using RT-qPCR and/or ELISA

• Signaling pathway analysis: Assess p38/JNK MAPK activation via phospho-specific antibodies

Research has demonstrated that TNFAIP1 coordinates with Cullin3 to mediate RhoB degradation, affecting inflammatory responses in cancer cells. Downregulation of TNFAIP1 induced the expression of pro-inflammatory cytokines IL-6 and IL-8 in TNFα-stimulated hepatocellular carcinoma cells through the activation of p38/JNK MAPK pathway .

What approaches can be used to study TNFAIP1's involvement in protein degradation pathways?

To investigate TNFAIP1's role in protein degradation:

• Protein stability assays:

  • Treat cells with cycloheximide (CHX) to block protein translation

  • Compare protein turnover rates in TNFAIP1-knockdown versus control cells

  • Analyze by Western blot at various time points (0-24h)

• Ubiquitination assays:

  • Transfect cells with HA-tagged ubiquitin and target protein

  • Immunoprecipitate the target protein using specific antibody

  • Detect ubiquitination by Western blot with anti-HA antibody

• E3 ligase complex formation:

  • Utilize pull-down assays with GST-TNFAIP1 purified from E. coli

  • Identify direct interactions with immunopurified His-tagged target proteins

  • Confirm interactions using co-immunoprecipitation and immunofluorescence co-localization

Research has shown that knockdown of TNFAIP1 significantly extended the half-life of RhoB in both Huh7 and HepG2 cells, while overexpression of HA-TNFAIP1 promoted RhoB degradation in a dose-dependent manner .

How can I address weak or non-specific signals when using TNFAIP1 antibodies in Western blotting?

When encountering issues with TNFAIP1 detection by Western blot, consider these optimization steps:

• Antibody selection: TNFAIP1 has a predicted molecular weight of 36 kDa. Ensure your antibody specifically recognizes this size band.

• Sample preparation:

  • Include protease inhibitors in lysis buffer

  • Avoid multiple freeze-thaw cycles

  • Consider enrichment of cellular fractions (cytoplasmic vs. membrane)

• Blocking optimization:

  • Test 5% non-fat milk vs. 3-5% BSA in TBST

  • For phospho-specific detection, BSA is preferred

• Antibody concentration:

  • For polyclonal antibodies: Try 1:300-1:1000 dilution range

  • For monoclonal antibodies: Try 1:1000-1:6000 dilution range

• Signal enhancement:

  • Extended exposure times

  • More sensitive detection systems (ECL Plus or Femto)

  • Consider loading more protein (50-80 μg)

If background remains high, try pre-adsorbing the antibody with cell lysate from a TNFAIP1-knockout cell line or adding 0.1% Tween-20 to the antibody dilution .

What are the critical parameters for successful immunohistochemical detection of TNFAIP1?

For optimal IHC results with TNFAIP1 antibodies:

• Fixation and processing:

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

  • Avoid over-fixation which can mask epitopes

• Antigen retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Heat-mediated retrieval is essential

• Antibody optimization:

  • For polyclonal antibodies: Test 1:20-1:200 dilution range

  • For monoclonal antibodies: Test 1:150-1:600 dilution range

  • Incubate overnight at 4°C for maximum sensitivity

• Detection system:

  • HRP-polymer systems generally provide better results than avidin-biotin methods

  • DAB development time is critical; monitor under microscope

• Validation controls:

  • Include positive control tissues (human placenta, brain)

  • Include negative controls (primary antibody omission)

  • Consider knockdown/knockout samples when available

TNFAIP1 shows both cytoplasmic and nuclear localization patterns, with tissue-specific expression patterns that should be considered when interpreting results .

How can TNFAIP1 antibodies be used to investigate its role in neuronal mitophagy and pyroptosis?

TNFAIP1 plays a critical role in neuronal function through SNAP25 regulation. To investigate this:

• Experimental design:

  • Use neuronal cell lines (SH-SY5Y, HT22, or primary neurons)

  • Create TNFAIP1 knockdown or overexpression models

  • Challenge with isoflurane+LPS to induce stress

• Key measurements:

  • Cell viability: MTT or CCK-8 assays

  • ROS detection: DCFH-DA fluorescence

  • Pyroptosis: Annexin V/PI double staining

  • Mitophagy markers: PINK1, Parkin, LC3-II, p62 (by Western blot)

  • SNAP25 expression levels

  • Caspase-3/GSDME cleavage

• Co-immunoprecipitation:

  • Use TNFAIP1 antibody to pull down associated proteins

  • Blot for SNAP25 to confirm interaction

  • Map interaction domains using truncation mutants

Research has demonstrated that TNFAIP1 negatively regulates SNAP25 protein abundance, which affects PINK1/Parkin-mediated mitophagy and caspase-3/GSDME-mediated pyroptosis. TNFAIP1 knockdown enhanced PINK1/Parkin-dependent mitophagy and inhibited pyroptosis in neuronal models .

What approaches can be used to study TNFAIP1's involvement in tumor suppression mechanisms?

TNFAIP1 has emerging roles as a tumor suppressor. To investigate:

• Expression analysis:

  • Compare TNFAIP1 levels in tumor vs. normal tissues using IHC (1:20-1:200 dilution)

  • Quantify differences in protein expression by Western blot

• Functional assays:

  • Proliferation: CCK8 assays with TNFAIP1 manipulation

  • Apoptosis: TUNEL assays and caspase activity measurements

  • Metastasis: Transwell migration and invasion assays

  • Angiogenesis: Tube formation assays with conditioned media

• Mechanistic studies:

  • Pathway analysis: Focus on NF-κB signaling

  • Protein-protein interactions: Co-IP with TNFAIP1 antibody followed by LC-MS/MS

  • Ubiquitination targets: Pull-down assays with TNFAIP1 and candidate proteins

• In vivo validation:

  • Xenograft models with TNFAIP1 overexpression/knockdown

  • IHC analysis of tumor sections for proliferation and apoptosis markers

Research has shown that TNFAIP1 suppresses hepatocellular carcinoma cell proliferation, induces apoptosis, and inhibits metastasis and angiogenesis both in vitro and in vivo .

How can recent findings on TNFAIP1's interactions with RhoB inform therapeutic strategies?

Recent research has established TNFAIP1 as a critical regulator of RhoB degradation with implications for inflammatory modulation:

• Therapeutic targeting strategies:

  • Small molecule inhibitors of TNFAIP1-RhoB interaction

  • Peptide-based interference of BTB domain function

  • RNA-based approaches to modulate TNFAIP1 expression

• Validation approaches:

  • In vitro binding assays with purified proteins

  • Cell-based assays measuring RhoB stability

  • Inflammatory cytokine profiling after modulation

• Readout measurements:

  • RhoB protein levels by Western blot

  • p38/JNK MAPK pathway activation

  • IL-6/IL-8 expression by RT-qPCR and ELISA

  • NF-κB signaling activity

Research has demonstrated that the TNFAIP1-RhoB axis provides a potential strategy for anti-inflammatory intervention in tumors. The mechanism involves Cullin3-TNFAIP1 E3 ligase controlling inflammatory response by mediating RhoB degradation through the ubiquitin proteasome system .

What are the considerations for developing novel TNFAIP1 antibodies for studying tissue-specific or isoform-specific functions?

For researchers developing specialized TNFAIP1 antibodies:

• Epitope selection considerations:

  • Target unique regions between species (for species-specific antibodies)

  • Focus on functional domains:

    • BTB domain (amino acids 1-96) for protein-protein interactions

    • N-terminal region (aa 27-52) shows high immunogenicity

  • Consider post-translational modifications that may affect function

• Validation requirements:

  • Parallel testing against knockout/knockdown samples

  • Cross-reactivity assessment with related proteins

  • Application-specific validation (WB, IHC, IP, ChIP)

  • Cell type-specific expression patterns

• Technical specifications:

  • For immunohistochemistry: Aim for 0.25-2 μg/mL working concentration

  • For WB detection: Target 1:1000 sensitivity range

  • For immunoprecipitation: ≥85% pull-down efficiency

• Special applications:

  • Super-resolution microscopy compatible antibodies

  • Live-cell imaging compatible (non-neutralizing)

  • ChIP-seq grade for transcriptional studies

When developing new antibodies, consider that TNFAIP1 antibodies targeting the N-terminal region (aa 27-52) have shown high specificity and functionality across multiple applications including Western blot, IHC, and ICC .