CNOT8 Antibody

What is CNOT8 and why is it important in cellular function?

CNOT8 (CCR4-NOT transcription complex subunit 8) is a critical component of the CCR4-NOT complex with 3'-5' poly(A) exoribonuclease activity. This 34 kDa protein plays essential roles in:

• Bulk mRNA degradation

• miRNA-mediated repression

• Translational repression during translational initiation

• General transcription regulation

• DNA damage response (DDR)

The protein is expressed ubiquitously and localizes to both the nucleus and cytoplasm. Research has shown that CNOT8 interacts with BTG family members like TOB1 and BTG2, mediating their anti-proliferative activity. CNOT8 appears to have partially redundant functions with CNOT7, another deadenylase subunit in the complex .

What types of CNOT8 antibodies are currently available for research applications?

Current research-grade CNOT8 antibodies include:

Most commercially available antibodies are rabbit polyclonals that recognize various epitopes of human CNOT8, with cross-reactivity to mouse CNOT8 in some cases .

What are the optimal dilutions for using CNOT8 antibodies in different applications?

Based on validated protocols, the following dilution ranges are recommended:

It is strongly recommended to titrate antibodies in each testing system to obtain optimal results as sensitivity can be sample-dependent .

How can I optimize immunofluorescence protocols when studying CNOT8 localization?

For optimal immunofluorescence detection of CNOT8:

• Fixation method: 4% paraformaldehyde at room temperature for 15 minutes has been validated for CNOT8 detection in HeLa cells .

• Antibody dilution: Begin with 1:200 dilution and adjust as needed; a range of 1:200-1:800 is typically effective .

• Nuclear counterstain: Hoechst 33342 provides good contrast with CNOT8 antibody signals .

• Visualization targets: Look for both nuclear and cytoplasmic staining, as CNOT8 localizes to both compartments.

• Controls: Include negative controls (secondary antibody only) and if possible, CNOT8 knockdown cells to validate specificity.

For HeLa cells specifically, antibody 10752-1-AP and NBP2-15930 have been validated to effectively detect CNOT8 protein localization .

How can CNOT8 antibodies be used to study DNA damage response pathways?

CNOT8 antibodies can be instrumental in investigating the role of CNOT8 in DNA damage response through several approaches:

• Phosphorylation state analysis: Western blotting with CNOT8 antibodies can be combined with phospho-specific antibodies against DDR proteins (γH2AX, RPA, 53BP1, and RAD51) to monitor changes following DNA damage induction.

• Foci formation quantification: Immunofluorescence microscopy using CNOT8 antibodies alongside antibodies against γH2AX, RPA, 53BP1, and RAD51 can quantify DNA damage foci formation. Research has shown that CNOT8 depletion resulted in significantly increased foci formation after 3 Gy ionizing radiation treatment, particularly at 8 hours post-irradiation .

• Co-localization studies: Use CNOT8 antibodies in conjunction with other DDR protein antibodies to examine potential co-localization at sites of DNA damage.

• Protocol considerations:

  • For irradiation experiments, expose cells to 3 Gy ionizing radiation

  • Collect samples at multiple timepoints (1, 2, 4, 8, and 24 hours post-irradiation)

  • Use CNOT8 siRNA-transfected cells as experimental group with control siRNA-transfected cells as comparison

Research demonstrates that CNOT8 depletion leads to cellular hypersensitivity to ionizing radiation, making CNOT8 antibodies valuable tools for investigating DDR pathways .

What are the technical challenges in using CNOT8 antibodies for distinguishing between CNOT7 and CNOT8 functions?

Distinguishing between CNOT7 and CNOT8 functions presents several technical challenges:

• Sequence homology: CNOT7 and CNOT8 are paralogues with significant sequence similarity, making antibody cross-reactivity a potential issue.

• Functional redundancy: Research indicates overlapping functions between CNOT7 and CNOT8, with knockdown of either resulting in significant differential expression of only a limited number of genes, suggesting compensatory mechanisms .

• Methodological approaches to overcome these challenges:

  • Epitope selection: Use antibodies targeting less conserved regions between CNOT7 and CNOT8

  • Validation strategy: Perform antibody validation using CNOT7 and CNOT8 knockout/knockdown cells to confirm specificity

  • Sequential immunoprecipitation: Use a two-step IP approach with CNOT7 depletion followed by CNOT8 antibody pulldown to identify unique interaction partners

  • Experimental design: Implement simultaneous knockdown of both proteins to identify truly redundant functions versus single knockdowns for protein-specific functions

• Controls: Include recombinant CNOT7 and CNOT8 proteins in Western blot analyses to verify antibody specificity and potential cross-reactivity .

How can I validate the specificity of a CNOT8 antibody before using it in critical experiments?

Comprehensive validation of CNOT8 antibodies should include:

• Western blot analysis:

  • Expected molecular weight: 34 kDa for human CNOT8

  • Positive controls: Jurkat cells, HeLa cells, and mouse testis tissue have shown detectable CNOT8 expression

  • Knockdown/knockout validation: Compare signal in wild-type versus CNOT8 siRNA-treated or CRISPR-edited cells

  • Multiple antibody comparison: Use at least two different antibodies targeting distinct epitopes

• Immunoprecipitation followed by mass spectrometry:

  • IP with CNOT8 antibody followed by MS identification confirms pulled-down protein is indeed CNOT8

  • Look for co-immunoprecipitation of known CNOT complex members

• Immunocytochemistry with knockdown controls:

  • Compare staining patterns in control versus CNOT8-depleted cells

  • Co-staining with antibodies against other CCR4-NOT complex components to confirm localization

• Recombinant protein testing:

  • Test antibody against purified recombinant CNOT8 protein

  • Include CNOT7 as a negative control to check for cross-reactivity with this paralog

• Cross-species validation:

  • If the antibody is predicted to recognize multiple species, confirm reactivity in each target species

  • Human and mouse CNOT8 show 99% sequence homology, making most antibodies cross-reactive

What are common problems encountered when using CNOT8 antibodies and how can they be resolved?

How can CNOT8 antibodies be effectively used in studying cell proliferation and viability?

Research has shown that CNOT8 depletion reduces cell viability, particularly at 72 hours post-siRNA transfection. To effectively use CNOT8 antibodies in proliferation studies:

• Experimental design considerations:

  • Timepoints: Monitor effects at 24h, 48h, 72h, and 96h post-intervention to capture the critical 72h timepoint where significant viability reduction is observed

  • Controls: Include both negative control siRNA and parallel CNOT7 siRNA treatments to assess redundancy

  • Cell types: HeLa cells have been validated, but consider testing multiple cell lines to assess tissue-specific effects

• Recommended methodology:

  • Cell viability: Use Cell Titer-Glo luminescent assay to measure ATP levels in CNOT8-depleted versus control cells

  • Antibody application: Monitor CNOT8 protein levels via Western blot (1:500-1:2000 dilution) to confirm knockdown efficiency

  • Complementary assays: Combine with BrdU incorporation and flow cytometry to assess cell cycle effects

• Data analysis considerations:

  • Perform statistical analysis to determine significance (p<0.05 considered significant)

  • Present data as relative luminescence units (RLU) normalized to control cells

  • Graph results with standard error bars from at least three independent experiments

What are the optimal protocols for using CNOT8 antibodies in RNA-protein interaction studies?

CNOT8's role in deadenylation and mRNA degradation makes RNA-protein interaction studies particularly valuable:

• RNA immunoprecipitation (RIP) protocol:

  • Cell lysis: Use NP-40 lysis buffer with RNase inhibitors and protease inhibitors

  • Pre-clearing: Pre-clear lysates with protein A/G beads for 1 hour at 4°C

  • Immunoprecipitation: Use 2-5 μg CNOT8 antibody per mg of protein lysate; incubate overnight at 4°C

  • RNA isolation: Extract RNA from immunoprecipitates using TRIzol or similar reagent

  • Analysis: Perform RT-qPCR or RNA-seq to identify bound transcripts

• Cross-linking immunoprecipitation (CLIP) considerations:

  • UV cross-linking (254 nm) to stabilize direct RNA-protein interactions

  • Include RNase treatment steps to trim RNA to footprint size

  • Use stringent wash conditions to reduce background

• Controls and validation:

  • Include IgG control immunoprecipitation

  • Validate with known CNOT complex-associated mRNAs

  • Confirm enrichment of deadenylated mRNAs

  • Compare RNA profiles from CNOT7 and CNOT8 immunoprecipitations to identify unique versus shared targets

• Technical considerations:

  • The recommended CNOT8 antibody concentration for immunoprecipitation is 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate

  • Mouse testis tissue has been validated for CNOT8 immunoprecipitation studies

How might CNOT8 antibodies contribute to understanding the role of CNOT8 in cancer therapeutics?

CNOT8 antibodies can facilitate several approaches to investigating CNOT8's potential role in cancer therapeutics:

• Expression profiling across cancer types:

  • Immunohistochemistry with CNOT8 antibodies can establish expression patterns across tumor types and stages

  • Western blot analyses can quantify expression levels in cancer cell lines versus normal counterparts

  • Correlate expression with patient outcomes to identify prognostic value

• Therapeutic target validation:

  • Use CNOT8 antibodies to monitor protein downregulation following therapeutic interventions

  • Combine with cell viability assays to correlate CNOT8 levels with treatment response

  • Immunoprecipitation studies can identify cancer-specific interaction partners as potential co-targets

• Mechanistic investigations:

  • Research has shown CNOT8 depletion increases sensitivity to DNA damaging agents, suggesting potential for combinatorial approaches

  • CNOT8 antibodies can monitor changes in localization and post-translational modifications following treatment

  • Co-immunoprecipitation studies can reveal alterations in CNOT complex composition in cancer contexts

• Biomarker development:

  • Standardized IHC protocols using validated CNOT8 antibodies could enable biomarker development

  • Recommended dilution ranges of 1:20-1:200 for IHC applications with antigen retrieval using TE buffer pH 9.0

Research indicates CNOT8 plays a role in cellular response to DNA damage and cell viability, making it a potential target for cancer therapeutic development, with CNOT8 antibodies serving as critical tools in this investigation .