ACTR10 Antibody

What is ACTR10 and why is it important in cancer research?

ACTR10 (Actin-related protein 10) is a component of the dynactin complex located at chromosome 14q23.1 with 13 exons. It plays critical roles in retrograde axonal transport of mitochondria and is present in the cytosol, extracellular region, and secretory granules . Recent research has identified ACTR10 as significantly upregulated in hepatocellular carcinoma (HCC) patients compared to non-tumor controls, with ACTR10 exerting pro-cancer effects by influencing RNA splicing, mRNA processing, and nucleocytoplasmic transport . Importantly, ACTR10 has emerged as an independent prognostic risk factor in HCC with a hazard ratio of 2.19 (95% CI: 1.56-3.08, P < 0.05) . Its involvement in tyrosine kinase inhibitor (TKI) resistance makes it a particularly valuable target for cancer research.

What validation methods should be employed when using ACTR10 antibodies?

When validating ACTR10 antibodies for research, implement a multi-step approach:

• Western blot validation: Compare protein detection in tissues/cells known to express ACTR10 (particularly HCC cell lines) versus negative controls

• siRNA/shRNA knockdown: Verify antibody specificity by confirming reduced signal following ACTR10 gene silencing

• Immunoprecipitation followed by mass spectrometry: Confirm the antibody pulls down authentic ACTR10 protein

• Immunofluorescence co-localization: Verify subcellular localization aligns with expected patterns (cytosol, mitochondrial associations, dynactin complex)

• Cross-reactivity testing: Ensure the antibody doesn't recognize related proteins like other actin-related family members

For particularly rigorous applications, CRISPR-Cas9 knockout cell lines provide the gold standard for antibody validation.

What experimental applications are most suitable for ACTR10 antibodies?

Selection should be guided by specific research questions, particularly when investigating TKI resistance mechanisms or dynactin complex interactions .

What are optimal storage and handling conditions for ACTR10 antibodies?

For maximum stability and performance of ACTR10 antibodies:

• Storage temperature: Store antibody aliquots at -20°C for long-term storage

• Aliquoting: Create single-use aliquots (10-50 μL) to avoid freeze-thaw cycles

• Carrier proteins: Ensure storage buffer contains stabilizers (BSA or glycerol)

• Working dilutions: Prepare fresh working dilutions on the day of experiment

• Temperature transitions: Allow antibodies to equilibrate to room temperature before opening tubes to prevent condensation

• Contamination prevention: Use sterile technique when handling antibody solutions

• Documentation: Maintain detailed records of lot numbers, dilutions, and experimental performance

These practices maximize reproducibility across experiments and extend antibody shelf-life.

How should researchers select between monoclonal and polyclonal ACTR10 antibodies?

For studies examining ACTR10's role in TKI resistance, monoclonal antibodies targeting specific functional domains may provide more consistent results across experiments .

What methodologies are most effective for studying ACTR10's role in TKI resistance?

ACTR10 has been validated as a TKI-resistance gene in HCC with a standardized mean difference (SMD) of 0.88 (95% CI: 0.01-0.76, P < 0.05) . Effective methodologies include:

• Co-immunoprecipitation with ACTR10 antibodies: To identify protein interactions changing during TKI resistance development

• Proximity labeling techniques: BioID or APEX2 fused to ACTR10 to map resistance-specific interaction networks

• Quantitative phosphoproteomics: Compare phosphorylation states of ACTR10 between TKI-sensitive and resistant cells

• Live-cell imaging with fluorescently tagged ACTR10: Track dynamics during resistance development

• ACTR10 overexpression/knockdown in patient-derived xenografts: Assess impact on TKI response in vivo

These approaches should focus on ACTR10's effects on exocytosis, autophagy, and apoptosis pathways, as enrichment analyses have identified these as key mechanisms in TKI resistance .

How can researchers troubleshoot inconsistent ACTR10 antibody performance?

When encountering inconsistent ACTR10 antibody results:

• Epitope masking: If the epitope is near protein interaction sites (particularly with dynactin complex components), try alternative extraction conditions or different antibody clones

• Expression level variations: Standardize loading by total protein rather than housekeeping genes, as ACTR10 expression varies significantly across cell types

• Cross-reactivity: If detecting unexpected bands, perform blocking experiments with recombinant ACTR10 peptides

• Post-translational modifications: Test dephosphorylation treatments if phosphorylation may be affecting epitope recognition

• Alternative fixation protocols: For immunohistochemistry/immunofluorescence, compare paraformaldehyde, methanol, and acetone fixation

• Sample preparation timing: ACTR10 may undergo rapid degradation; standardize time from sample collection to processing

When investigating ACTR10 in TKI resistance contexts, standardize cell treatment conditions, as ACTR10 expression profiles change significantly between TKI-sensitive and resistant samples .

What approaches are recommended for investigating ACTR10's interactions with the dynactin complex?

ACTR10 is a component of the dynactin complex, with research showing it can bind mitochondria even when lacking its dynactin binding domain . Recommended approaches include:

• Sequential co-immunoprecipitation: First pull down with dynactin components, then with ACTR10 antibodies to identify direct vs. indirect interactions

• Domain-specific antibodies: Use antibodies targeting different ACTR10 domains to map interaction interfaces

• In situ proximity ligation assay: Visualize ACTR10-dynactin component interactions in their native cellular context

• Cross-linking mass spectrometry: Identify precise binding interfaces between ACTR10 and other complex components

• FRET/FLIM microscopy: Measure real-time interactions in living cells using fluorescently tagged components

These approaches help elucidate how ACTR10 contributes to dynactin function in mitochondrial transport and other cellular processes.

What strategies can improve ACTR10 antibody performance in challenging tissue samples?

For difficult tissue samples, particularly formalin-fixed paraffin-embedded (FFPE) HCC specimens:

• Optimized antigen retrieval: Test both heat-induced (citrate, EDTA, Tris buffers at varying pH) and enzymatic methods

• Signal amplification systems: Employ tyramide signal amplification or polymer-based detection systems

• Extended primary antibody incubation: Consider overnight incubation at 4°C to improve penetration

• Background reduction: Use specialized blocking solutions containing both proteins and detergents

• Tissue section thickness optimization: Compare 3-5μm sections for optimal signal-to-noise ratio

• Dual antibody approach: Use two different ACTR10 antibodies targeting distinct epitopes

• Automated staining platforms: Standardize staining conditions across batches

These approaches are particularly valuable when studying ACTR10 in HCC tissues to assess its prognostic significance, as ACTR10 has been identified as an independent prognostic risk factor .

How can ACTR10 antibodies be employed in studying autophagy and exocytosis mechanisms?

ACTR10 mediates TKI resistance through enhanced exocytosis, autophagy, and apoptosis in HCC patients . To investigate these mechanisms:

For autophagy studies:

• Co-localization with LC3B: Dual immunofluorescence with ACTR10 and autophagy markers

• Autophagosome isolation: Use ACTR10 antibodies to assess enrichment in isolated autophagosomes

• Autophagic flux assays: Compare ACTR10 localization before/after bafilomycin A1 treatment

• Proximity labeling: Identify autophagy-specific ACTR10 interactors during TKI resistance

For exocytosis studies:

• SNARE protein interactions: Co-immunoprecipitation of ACTR10 with SNARE proteins

• Live-cell imaging: Track ACTR10-positive vesicles during exocytosis events

• Calcium-dependent regulation: Assess ACTR10 phosphorylation state changes during calcium-triggered exocytosis

• Super-resolution microscopy: Visualize ACTR10 at exocytic vesicle fusion sites

GO analysis has identified ACTR10's involvement in positive regulation of exocytosis, membrane docking, and autophagy pathways, making these processes critical targets for understanding TKI resistance mechanisms .