ACT1 Antibody

What is ACT1 and what are its primary functions in immune signaling?

ACT1 functions as a key adaptor molecule in IL-17 receptor signaling and plays crucial roles in both transcriptional and posttranscriptional regulation of proinflammatory genes. It serves as a scaffold protein facilitating interaction between IKKγ and other signaling molecules, promoting activation of NFκB and JNK pathways in response to proinflammatory cytokines such as IL-1 and TNFα . Paradoxically, ACT1 also functions as a negative regulator in T and B cells through direct inhibition of STAT3, making it an intricate modulator of immune responses . The protein contains a C-terminal SEFIR domain that mediates association with IL-17R and an N-terminal region (first 15 amino acids) that interacts with TRAF6, separated by a disordered mid-region subject to regulatory phosphorylation .

How does ACT1 deficiency affect immune function in research models?

ACT1 deficiency results in hyperactivated follicular Th17 cells with elevated IL-21 expression, which promotes T-B cell interaction leading to B cell expansion and aberrant antibody production . Mice lacking ACT1 develop systemic autoimmunity resembling Systemic Lupus Erythematosus (SLE) and Sjögren's-like diseases, characterized by hypergammaglobulinemia, elevated serum autoantibodies, and organ-specific inflammation . Specifically, ACT1-deficient mice show hyper IL-23-induced STAT3 activation in naive CD4+ T cells and increased IL-21 expression, demonstrating that ACT1's negative regulatory function is critical for preventing autoimmunity .

What criteria should researchers consider when selecting an ACT1 antibody for their experiments?

When selecting an ACT1 antibody, researchers should consider the following parameters:

• Species reactivity: Verify the antibody detects ACT1 in your species of interest (e.g., mouse, rat, human).

• Applications compatibility: Ensure compatibility with your intended application (WB, IP, IF, ELISA).

• Domain specificity: Select antibodies targeting relevant domains based on your research questions (SEFIR domain vs N-terminal region).

• Validation data: Review existing literature demonstrating the antibody's specificity and performance.

• Conjugation needs: Determine if you require unconjugated antibody or specific conjugates (HRP, fluorophores) based on your detection method .

The D-11 mouse monoclonal ACT1 antibody, for example, detects ACT1 protein from mouse, rat, and human origins and is validated for western blotting, immunoprecipitation, immunofluorescence, and ELISA applications .

How should researchers validate ACT1 antibody specificity in their experimental systems?

Validation of ACT1 antibody specificity should include:

• Positive and negative controls: Use samples with known ACT1 expression levels, including ACT1-deficient cells (Act1−/−) as negative controls.

• Multiple detection methods: Confirm results using at least two different techniques (e.g., WB and IF).

• Knockdown/knockout validation: Demonstrate reduced or absent signal in ACT1 knockdown or knockout samples.

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to demonstrate specific binding.

• Molecular weight verification: Confirm detection at the expected molecular weight (approximately 60 kDa).

For immunoprecipitation experiments, researchers can validate results by detecting ACT1-associated proteins known to interact with it, such as IL-17R, TRAF6, or STAT3 .

How can researchers optimize ACT1 antibody usage for CLIP-seq experiments to study ACT1-RNA interactions?

For CLIP-seq experiments investigating ACT1-RNA interactions, researchers should:

• Cell preparation: Use cells stably expressing FLAG-tagged ACT1 to facilitate immunoprecipitation, as demonstrated in previous studies .

• Crosslinking optimization: Perform UV crosslinking at 254 nm to covalently link protein-RNA complexes while maintaining specificity.

• RNase treatment calibration: Titrate RNase concentration to generate RNA fragments of optimal size (30-50 nucleotides).

• Stringent washing conditions: Implement high-stringency washes to remove non-specific interactions.

• Library preparation: Generate and sequence libraries from isolated RNA fragments using standard RNA-seq protocols.

This approach has successfully identified that ACT1 binds in high density to the 5'UTR of specific transcripts including antioxidant mRNAs and WTAP (Wilms' tumor 1-associating protein), revealing its role in m6A RNA modification and redox homeostasis .

What protocols are recommended for studying ACT1 phosphorylation status in the IL-17 receptor signaling complex?

To study ACT1 phosphorylation within the IL-17 receptor signaling complex:

• Stimulation conditions: Treat cells with recombinant IL-17 (50-100 ng/ml) for short time periods (5-30 minutes).

• Lysis buffer optimization: Use buffers containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate) to preserve phosphorylation status.

• Immunoprecipitation strategy: Immunoprecipitate using antibodies against ACT1 or the IL-17 receptor.

• Phosphorylation detection: Analyze samples using:

  • Phospho-specific antibodies if available

  • Mass spectrometry to identify specific phosphorylation sites

  • Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated forms

Research has shown that ACT1 in the IL-17 receptor signaling complex is phosphorylated at multiple sites within its unstructured mid-region, and these phosphorylation events regulate TRAF6 recruitment and downstream signaling .

How can ACT1 antibodies be utilized to investigate the mechanistic link between ACT1 mutations and autoimmune diseases?

To investigate the relationship between ACT1 mutations and autoimmune diseases:

• Genetic screening: Use ACT1 antibodies in combination with genetic analysis to identify patient populations with ACT1 variants, particularly the SNP-D10N mutation linked to psoriasis and SLE.

• Structure-function analysis: Compare wild-type and mutant ACT1 protein interactions using co-immunoprecipitation with ACT1 antibodies to identify altered binding partners.

• Signaling pathway analysis: Assess STAT3 activation levels in cells expressing wild-type versus mutant ACT1 using phospho-STAT3 antibodies alongside ACT1 antibodies.

• Animal models: Generate knock-in models with specific ACT1 mutations and use ACT1 antibodies to track protein expression and localization.

• Therapeutic screening: Utilize ACT1 antibodies to monitor effects of potential therapeutics, such as IL-21 blocking antibodies which have shown efficacy in ameliorating SLE- and Sjögren's-like diseases in Act1-deficient mice .

What methodological approaches can be used to study ACT1's dual function in both promoting and inhibiting immune responses?

To investigate ACT1's contextual roles:

• Domain-specific antibodies: Use antibodies targeting distinct functional domains of ACT1 (N-terminal TRAF6-binding region versus C-terminal SEFIR domain) to distinguish pathway-specific interactions.

• Cell-type specific analysis: Compare ACT1 functions across different immune cell types (T cells, B cells, fibroblasts) using flow cytometry with ACT1 antibodies and lineage markers.

• Temporal signaling studies: Track ACT1-dependent signaling at different time points after stimulation using phospho-specific antibodies for downstream effectors.

• Reconstitution experiments: Complement ACT1-deficient cells with wild-type or mutant ACT1 constructs (e.g., ACT1 Δ20-380 or phosphorylation site mutants) and analyze signaling outcomes .

• Proximity labeling: Employ BioID or APEX2-based approaches with ACT1 fusion proteins to identify context-specific interaction partners.

Research has demonstrated that mutation of all phosphorylation sites in ACT1's disordered mid-region substantially increases signaling responses to IL-17 and enhances TRAF6 recruitment, revealing how post-translational modifications regulate ACT1's signaling functions .

What are common technical challenges when using ACT1 antibodies and recommended solutions?

How should researchers interpret contradictory data regarding ACT1 function in different experimental contexts?

When confronting contradictory findings about ACT1 function:

• Context consideration: Assess whether differences are due to cell type-specific effects. For example, ACT1 deficiency has distinct consequences in T cells versus other cell types .

• Pathway-specific analysis: Determine if contradictions relate to specific pathways. ACT1 can both mediate IL-17 signaling and negatively regulate STAT3, creating apparently opposing functions .

• Domain functionality: Analyze which functional domain of ACT1 is relevant to your experiment. The N-terminal TRAF6-binding region and C-terminal SEFIR domain mediate different interactions .

• Genetic background effects: Consider strain-specific differences if using animal models. The autoimmune phenotype in ACT1-deficient mice has been well-documented in Balb/c background .

• Phosphorylation status: Evaluate the phosphorylation state of ACT1, as this dramatically affects its signaling properties. Mutation of all phospho-sites substantially increases signaling responses to IL-17 .

How might ACT1 antibodies be applied to study its emerging role in cancer chemoresistance?

Recent research has revealed that IL-17/ACT1 induces chemoresistance by modulating redox homeostasis through epitranscriptomic regulation of antioxidant RNA metabolism . To investigate this emerging role:

• Translational regulation analysis: Use ACT1 antibodies in RNA immunoprecipitation (RIP) assays to capture and identify mRNAs bound by ACT1 in chemoresistant versus chemosensitive cells.

• Epitranscriptomic modifications: Combine ACT1 antibodies with m6A-specific antibodies to study how ACT1 affects RNA methylation patterns that contribute to chemoresistance.

• Redox homeostasis pathway: Employ ACT1 antibodies alongside redox-sensitive probes to monitor how ACT1 status affects cellular redox balance in response to chemotherapy.

• Therapeutic targeting: Use ACT1 antibodies to screen for compounds that disrupt ACT1-RNA interactions or ACT1-dependent signaling pathways as potential chemosensitizers.

• Patient sample analysis: Apply ACT1 immunohistochemistry to cancer patient samples to correlate ACT1 expression levels with treatment response and survival outcomes.

What experimental approaches could elucidate the mechanisms by which ACT1 directly binds and regulates inflammatory mRNAs?

To study ACT1's RNA-binding activities:

• Structure-function analysis: Use domain-specific ACT1 antibodies to determine which regions mediate RNA binding, particularly focusing on the SEFIR domain previously implicated in stem-loop RNA binding .

• RNA structure determination: Combine ACT1 immunoprecipitation with RNA structure probing techniques to identify specific RNA motifs or structures recognized by ACT1.

• In vitro binding assays: Perform electrophoretic mobility shift assays (EMSAs) with purified ACT1 protein and synthetic RNA oligonucleotides to quantify binding affinities.

• Live-cell imaging: Develop fluorescently tagged ACT1 and RNA systems to visualize dynamic interactions in real-time during inflammatory responses.

• Targeted mutagenesis: Generate ACT1 mutants with altered RNA-binding properties and assess their effects on inflammatory gene expression and mRNA stability.