ACTR5 Antibody

What is ACTR5 and what are its primary cellular functions?

ACTR5 (Actin-related protein 5) is a proposed core component of the chromatin remodeling INO80 complex involved in multiple cellular processes including transcriptional regulation, DNA replication, and DNA repair mechanisms. It plays a crucial role in DNA double-strand break repair and UV-damage excision repair . Recent research has identified ACTR5 as essential for hepatocellular carcinoma (HCC) tumor progression, where it functions in CDKN2A silencing and controls CDK6/E2F1-mediated cell cycle progression . The protein represents a link between chromatin dynamics and cellular genome maintenance mechanisms .

What types of ACTR5 antibodies are available for research?

Based on current research resources, polyclonal antibodies against ACTR5 are commonly used in laboratory settings. For example, Rabbit Polyclonal ACTR5 antibodies have been developed that recognize immunogens corresponding to recombinant fragment proteins within Human ACTR5 (particularly amino acids 150-300) . These antibodies are suitable for multiple applications including immunohistochemistry on paraffin-embedded tissues (IHC-P) and immunocytochemistry/immunofluorescence (ICC/IF), and have been validated to react with human samples .

What are the recommended applications for ACTR5 antibodies?

ACTR5 antibodies have been validated for several research applications:

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) - Useful for detecting ACTR5 expression in fixed tissue samples, as demonstrated in human kidney and liver tissues

• Immunocytochemistry/immunofluorescence (ICC/IF) - Effective for cellular localization studies, validated in human liver hepatocellular carcinoma cell lines such as HepG2

• ChIP-seq experiments - ACTR5 antibodies can be used for chromatin immunoprecipitation followed by sequencing to identify genomic binding sites

When selecting an application, researchers should consider the specific experimental question, sample type, and available validation data for the antibody in the relevant application and species.

How should I optimize immunohistochemistry protocols for ACTR5 detection?

For optimal ACTR5 detection in immunohistochemistry on paraffin-embedded tissues:

• Antibody dilution: Start with a 1/100 dilution of the primary antibody as a baseline, which has been validated for human tissue samples including kidney and liver

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) before antibody incubation

• Incubation conditions: Incubate with primary antibody overnight at 4°C to maximize specific binding

• Detection system: Use appropriate secondary antibodies conjugated with horseradish peroxidase or fluorophores depending on the desired visualization method

• Controls: Always include positive control tissues (such as human liver) and negative controls (primary antibody omission) to validate staining specificity

The protocol may require further optimization based on specific tissue types and fixation methods. Monitoring background levels and signal-to-noise ratio will help determine optimal conditions for your specific experimental setup.

What is the recommended protocol for ACTR5 detection by immunofluorescence?

For immunofluorescence detection of ACTR5:

• Cell preparation: Culture cells (e.g., HepG2) on glass coverslips and fix with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: Treat with 0.1% Triton X-100 for 10 minutes to allow antibody access to intracellular targets

• Blocking: Incubate with 5% normal serum in PBS for 1 hour to reduce non-specific binding

• Primary antibody: Apply ACTR5 antibody at 1/100 dilution and incubate overnight at 4°C

• Secondary antibody: Use fluorophore-conjugated secondary antibodies such as Alexa Fluor 488® conjugated Goat Anti-Rabbit IgG at 1/500 dilution for 1 hour at room temperature

• Nuclear counterstain: Apply DAPI (1 μg/mL) for 5 minutes to visualize nuclei

• Mounting: Mount slides with anti-fade mounting medium and seal edges

This protocol has been validated for human liver hepatocellular carcinoma cell lines and provides clear visualization of ACTR5 subcellular localization .

How can ACTR5 antibodies be utilized in chromatin immunoprecipitation (ChIP) experiments?

For ACTR5 ChIP experiments:

• Crosslinking: Treat cells with 1% formaldehyde for 10 minutes to crosslink protein-DNA complexes

• Chromatin preparation: Lyse cells, isolate nuclei, and sonicate chromatin to fragments of 200-500 bp

• Immunoprecipitation: Incubate sonicated chromatin with ACTR5 antibody overnight at 4°C

• Bead capture: Add protein A/G magnetic beads for 2 hours at 4°C to capture antibody-protein-DNA complexes

• Washes: Perform stringent washes to remove non-specific binding

• Elution and crosslink reversal: Elute complexes and reverse crosslinks at 65°C overnight

• DNA purification: Purify DNA for downstream analysis by qPCR or sequencing

Research has demonstrated that ACTR5 binds to the CDKN2A promoter region, which can be confirmed by both ChIP-seq and ChIP-qPCR approaches . When designing ChIP experiments, specific attention should be paid to the regulatory regions of cell cycle-related genes, particularly those in the E2F pathway, as ACTR5 has been shown to bind approximately 525 genes with substantial overlap with E2F pathway genes .

What is the role of ACTR5 in regulating the cell cycle, and how can antibodies help investigate this function?

ACTR5 plays a crucial role in cell cycle regulation through its involvement in CDKN2A silencing and subsequent effects on the CDK6/E2F1 pathway. Research findings demonstrate:

• CDKN2A regulation: ACTR5 suppression leads to induction of CDKN2A mRNA and protein expression

• Downstream effects: ACTR5 knockdown causes reduction of CDK6, phospho-S780 Rb (p-Rb), and E2F1 protein levels in ACTR5-dependent HCC cells

• Cell cycle impact: ACTR5 suppression significantly reduces cells in S phase

To investigate these functions:

• Use ACTR5 antibodies for ChIP-seq to identify binding sites at cell cycle regulatory genes

• Combine with immunoblotting to examine effects on downstream proteins (CDK6, p-Rb, E2F1)

• Correlate with flow cytometry analysis of cell cycle distribution

• Compare effects in ACTR5-dependent and ACTR5-independent cell lines

This multilayered approach can provide comprehensive insights into ACTR5's role in cell cycle regulation across different cellular contexts.

How do ACTR5 antibodies perform in protein complex immunoprecipitation for studying INO80 complex interactions?

For studying ACTR5's interactions within and outside the INO80 complex:

• Lysis conditions: Use mild detergent buffers (e.g., 0.5% NP-40) to preserve protein-protein interactions

• Pre-clearing: Incubate lysates with protein A/G beads to reduce non-specific binding

• Immunoprecipitation: Incubate with ACTR5 antibody coupled to protein A/G beads overnight at 4°C

• Washes: Perform gentle washes to maintain complex integrity

• Elution: Use gentle elution methods to preserve protein complexes

• Analysis: Analyze by western blot or mass spectrometry (LC-MS/MS)

Recent research indicates that ACTR5 interacts with IES6 (INO80 complex subunit C), which is crucial for stabilizing ACTR5 protein and maintaining HCC proliferation . Interestingly, studies suggest an INO80-independent mechanism of ACTR5/IES6 in supporting HCC proliferation . When designing co-immunoprecipitation experiments, researchers should consider examining both canonical INO80 complex members (INO80, MCRS1, ACTR8, and YY1) and potential INO80-independent interactions to fully understand ACTR5's functional roles.

What are common issues with ACTR5 antibody specificity and how can they be addressed?

Common specificity issues with ACTR5 antibodies include:

• Cross-reactivity: ACTR5 belongs to the actin-related protein family, which shares structural similarities with other family members

• Batch-to-batch variation: Particularly with polyclonal antibodies

• Non-specific binding: Can lead to false-positive signals

To address these issues:

Always validate antibody specificity using multiple approaches, including western blot, immunoprecipitation followed by mass spectrometry, and ideally genetic knockdown or knockout controls to confirm signal specificity.

How can I validate ACTR5 antibody performance for my specific experimental system?

A comprehensive validation approach for ACTR5 antibodies includes:

• Expression correlation: Compare antibody signal with known ACTR5 expression levels across different cell types

• Genetic manipulation: Test antibody in CRISPR knockdown/knockout systems (e.g., using CRISPRi with sgRNAs targeting ACTR5)

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm epitope specificity

• Application-specific validation:

  • For western blot: Confirm single band at expected molecular weight

  • For IHC/ICC: Compare staining pattern with published subcellular localization

  • For ChIP: Perform ChIP-qPCR at known binding sites (e.g., CDKN2A promoter)

When working with new cell types or tissues, researchers should first establish baseline ACTR5 expression using quantitative PCR or publicly available expression databases before proceeding with antibody-based detection.

How can ACTR5 antibodies be used to investigate the INO80-independent functions of ACTR5/IES6?

To investigate the INO80-independent functions of ACTR5/IES6:

• Differential co-immunoprecipitation: Use ACTR5 antibodies to pull down protein complexes under different cellular conditions (e.g., normal vs. cancer cells) and identify differential interactors by mass spectrometry

• Proximity labeling: Combine ACTR5 antibodies with proximity labeling techniques (BioID or APEX) to identify context-specific interactors

• ChIP-seq comparative analysis: Compare ACTR5 binding sites with those of core INO80 components to identify unique ACTR5 targets

• Domain-specific antibodies: Develop antibodies targeting specific ACTR5 domains identified through CRISPR tiling screens to distinguish between INO80-dependent and independent functions

Research has revealed a distinct HCC-specific usage of ACTR5 and IES6 compared to other INO80 complex members, suggesting an INO80-independent mechanism in supporting HCC proliferation . This distinction was identified through high-density CRISPR gene tiling scans, highlighting the importance of domain-specific analysis in understanding ACTR5 function .

What are the emerging applications of ACTR5 antibodies in cancer research and potential therapeutic development?

Emerging applications of ACTR5 antibodies in cancer research include:

• Prognostic biomarker development: High ACTR5 expression correlates with poor survival prognosis in HCC patients

• Therapeutic target validation: ACTR5 suppression significantly retards in vivo HCC tumor progression and reduces tumor mass (demonstrated reduction from 0.537±0.097g to 0.013±0.006g in HepG2 xenograft models)

• Mechanism elucidation: ACTR5 antibodies help reveal how ACTR5 controls CDKN2A expression through changes in histone modifications (H3K9me2 and H3K27me3)

• Patient stratification: Potential use in identifying patients who might benefit from therapies targeting ACTR5-dependent pathways

For therapeutic development, researchers can use ACTR5 antibodies to:

• Screen for compounds that disrupt ACTR5-IES6 interaction

• Validate on-target effects of potential ACTR5 inhibitors

• Monitor changes in ACTR5 expression or localization following treatment

• Identify synergistic therapeutic combinations targeting ACTR5-dependent pathways

How can multiplexed antibody approaches be optimized for studying ACTR5 in the context of chromatin remodeling complexes?

For optimized multiplexed antibody approaches:

• Antibody panel selection: Combine ACTR5 antibodies with antibodies against:

  • Core INO80 complex members (INO80, ACTR8, MCRS1, YY1)

  • IES6/INO80C (direct ACTR5 partner)

  • Histone modifications affected by ACTR5 (H3K9me2, H3K27me3)

  • Cell cycle regulators (CDKN2A, CDK6, E2F1, phospho-Rb)

• Multiplexed imaging techniques:

  • Sequential immunofluorescence with antibody stripping

  • Mass cytometry (CyTOF) for single-cell protein quantification

  • Imaging mass cytometry for spatial context

  • Cyclic immunofluorescence (CycIF) for co-localization studies

• Controls and normalization:

  • Use spike-in controls for batch correction

  • Include channel crossover controls for spectral unmixing

  • Apply computational approaches to correct for antibody affinity differences

This multiplexed approach can provide comprehensive insights into the dynamic composition and function of ACTR5-containing complexes across different cellular states and chromatin contexts, revealing how ACTR5 contributes to both INO80-dependent and independent functions in normal and cancer cells.