CNOT4 Antibody

Basic Research Questions

• What is CNOT4 and what biological functions does it serve?

CNOT4 is a 575 amino acid protein with E3 ubiquitin ligase activity, primarily involved in protein modification and ubiquitination. It contains a RING type zinc finger domain of the C4C4 type, an RNA recognition motif, and a bipartite nuclear localization signal. As a component of the CCR4-NOT core complex, CNOT4 functions in general transcription regulation with both positive and negative effects on the transcription of multiple functionally unrelated genes. It interacts with CNOT1 via its C-terminus and with E2 ubiquitin ligases via its C4C4 RING domain .

CNOT4 is involved in the activation of the JAK/STAT pathway and can get phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR . Additionally, it plays a role in quality control of translation of mitochondrial outer membrane-localized mRNA and, as part of PINK1-regulated signaling, ubiquitinates ABCE1 upon mitochondria damage to initiate mitophagy .

• What types of CNOT4 antibodies are available for research applications?

Several types of CNOT4 antibodies are available for research purposes, each with specific characteristics:

When selecting a CNOT4 antibody, researchers should consider the specific application, target species, and required epitope region based on their experimental design .

• What is the recommended protocol for using CNOT4 antibodies in Western Blotting?

For Western Blotting applications with CNOT4 antibodies, follow these methodological steps:

• Sample preparation: Extract proteins from your sample of interest using an appropriate lysis buffer that preserves protein integrity.

• Protein quantification: Determine protein concentration using Bradford or BCA assay.

• SDS-PAGE: Load 20-50 μg of protein per lane on a 10% SDS-PAGE gel.

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane.

• Blocking: Block the membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute CNOT4 antibody according to manufacturer recommendations (typically 1:200-1:1000 for polyclonal antibodies or specified dilution for monoclonals) .

• Secondary antibody incubation: Use appropriate HRP-conjugated secondary antibody.

• Detection: Develop using ECL substrate and visualize bands.

Note that CNOT4 is typically observed at 64-85 kDa molecular weight, which may vary depending on post-translational modifications . Include positive controls such as mouse skeletal muscle tissue, MCF-7 cells, or mouse testis tissue, which have shown positive WB results .

• How should I prepare samples for CNOT4 immunohistochemistry?

For optimal CNOT4 immunohistochemistry results, follow these methodological guidelines:

• Tissue fixation: Fix tissues in 10% neutral buffered formalin for 24-48 hours.

• Processing and embedding: Process and embed tissues in paraffin following standard protocols.

• Sectioning: Cut 4-6 μm thick sections and mount on positively charged slides.

• Deparaffinization and rehydration: Use xylene and descending grades of ethanol.

• Antigen retrieval: This is critical for CNOT4 detection. Use TE buffer pH 9.0 as recommended, or alternatively, citrate buffer pH 6.0 .

• Endogenous peroxidase blocking: Incubate in 3% hydrogen peroxide for 10 minutes.

• Antibody dilution: Dilute CNOT4 antibody at 1:20-1:200 as recommended .

• Incubation: Incubate primary antibody overnight at 4°C.

• Detection: Use an appropriate detection system compatible with the host species of your primary antibody.

Human lung cancer tissue has shown positive IHC results, and human cerebellum exhibits moderate to strong cytoplasmic positivity in Purkinje cells .

Advanced Research Questions

• How does CNOT4 expression influence cancer progression and immunotherapy response?

In experimental models, CNOT4 overexpression suppresses tumor growth in vivo and significantly enhances the efficacy of anti-PD-1 immunotherapy . The mechanism appears to involve increased infiltration of CD3+ and CD8+ cytotoxic T lymphocytes into the tumor microenvironment, as demonstrated in studies using A549 cell line-derived tumors in mice .

When CNOT4 is overexpressed in combination with anti-PD-1 treatment, researchers observed:

• Markedly enhanced fractions of CD3+ and CD8+ T lymphocytes in tumors

• Significantly increased expression of lymphokines, including TNF-α and IFN-γ

• Greater tumor growth suppression compared to either intervention alone

This suggests CNOT4 could serve as both a prognostic marker for NSCLC and a potential combinational target with anti-PD-1 treatment for NSCLC patients .

• What molecular mechanisms underlie CNOT4's role in enhancing anti-PD-1 immunotherapy?

The mechanisms by which CNOT4 enhances anti-PD-1 immunotherapy involve complex immunological processes:

• Immune cell recruitment: CNOT4 overexpression increases the infiltration of CD3+ and CD8+ cytotoxic T lymphocytes into tumors, which are critical for anti-tumor immunity .

• Lymphokine upregulation: CNOT4 promotes increased expression of key immune-stimulatory cytokines:

  • TNF-α expression is enhanced after CNOT4 overexpression and further amplified when combined with anti-PD-1 treatment

  • IFN-γ is similarly upregulated, with the highest expression observed in the combination therapy group

• Synergistic effect with checkpoint blockade: The PD-1/PD-L1 axis normally suppresses T cell activation and proliferation. CNOT4 appears to work synergistically with anti-PD-1 treatment to overcome this immune suppression, resulting in enhanced cytotoxic T cell activity .

• Potential molecular interactions: While the precise molecular mechanisms remain under investigation, CNOT4's E3 ubiquitin ligase activity may be involved in regulating proteins that influence immune response pathways .

This research suggests that combining CNOT4-targeting strategies with immune checkpoint inhibitors could represent a promising approach for improving immunotherapy outcomes, particularly in NSCLC patients .

• How can I optimize CNOT4 antibody validation to ensure experimental reliability?

Rigorous validation of CNOT4 antibodies is essential for experimental reliability. Follow these methodological approaches:

• Positive and negative controls:

  • Positive tissue controls: Use tissues known to express CNOT4, such as mouse skeletal muscle, testis, or human lung cancer tissue .

  • Cell line controls: MCF-7 and HepG2 cells have shown positive results for CNOT4 detection .

  • Negative controls: Include appropriate negative controls (omit primary antibody, use isotype control, or use tissues known not to express CNOT4).

• Specificity verification methods:

  • Knockdown/knockout validation: Use CNOT4 siRNA or CRISPR-Cas9 knockout cells to confirm antibody specificity.

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

  • Multiple antibody approach: Use antibodies targeting different epitopes of CNOT4 and compare results.

• Cross-reactivity assessment:

  • Test the antibody across multiple species if cross-reactivity is claimed (human, mouse, rat) .

  • Verify expected molecular weight (64-85 kDa for CNOT4) .

  • Compare staining patterns across different applications (WB, IHC, IF) for consistency.

• Application-specific validation:

  • For Western blotting: Verify single band at expected molecular weight.

  • For IHC/IF: Confirm expected subcellular localization (CNOT4 shows cytoplasmic positivity in Purkinje cells) .

• What technical considerations are important when interpreting CNOT4 localization in immunofluorescence studies?

When interpreting CNOT4 localization in immunofluorescence studies, several technical considerations are crucial:

• Subcellular localization patterns:

  • CNOT4 contains a bipartite nuclear localization signal but has been observed to show cytoplasmic localization in certain cell types .

  • In Purkinje cells, for example, CNOT4 shows moderate to strong cytoplasmic positivity .

  • The localization may vary depending on cell type, physiological state, and experimental conditions.

• Fixation and permeabilization influence:

  • Different fixation methods (paraformaldehyde, methanol, acetone) can affect epitope accessibility and apparent localization.

  • Permeabilization conditions can influence antibody penetration and staining patterns.

  • Test different fixation/permeabilization combinations to optimize CNOT4 detection.

• Antibody selection considerations:

  • Different antibodies (recognizing different epitopes) may reveal different localization patterns.

  • Monoclonal antibodies might miss certain epitopes due to conformational changes or protein interactions.

  • Polyclonal antibodies provide broader epitope recognition but may show higher background.

• Co-localization studies:

  • Use markers for specific cellular compartments (nucleus, cytoplasm, mitochondria) to precisely determine CNOT4 localization.

  • Consider that CNOT4's role in quality control of mitochondrial outer membrane-localized mRNA translation suggests potential mitochondrial association .

  • CNOT4's interaction with the CCR4-NOT complex may influence its localization in RNA processing bodies.

• Functional state considerations:

  • CNOT4 phosphorylation in response to DNA damage may alter its localization .

  • Different experimental conditions (stress, growth factors, cell cycle stage) might affect CNOT4 distribution.

  • Consider time-course experiments to capture dynamic changes in localization.

For optimal results in HepG2 cells, which have shown positive IF/ICC results, use a dilution range of 1:10-1:100 for CNOT4 antibodies .

• How can CNOT4 antibodies be applied to investigate its role in the ubiquitin-proteasome system?

CNOT4's E3 ubiquitin ligase activity makes it an important player in the ubiquitin-proteasome system. CNOT4 antibodies can be applied in several experimental approaches to investigate this role:

• Immunoprecipitation (IP) followed by ubiquitination assays:

  • Use CNOT4 antibodies to immunoprecipitate CNOT4 and its interacting proteins.

  • Perform Western blotting with anti-ubiquitin antibodies to detect ubiquitinated proteins.

  • This approach can identify potential CNOT4 substrates and study ubiquitination patterns (mono- vs. poly-ubiquitination).

• Co-immunoprecipitation to identify interaction partners:

  • IP with CNOT4 antibodies followed by mass spectrometry can identify novel CNOT4-interacting proteins.

  • Verify interactions with specific Western blotting for known E2 ubiquitin-conjugating enzymes.

  • The search results indicate that CNOT4 interacts with E2 ubiquitin ligases via its C4C4 RING domain .

• In vitro ubiquitination assays:

  • Immunopurify CNOT4 using specific antibodies for use in reconstituted ubiquitination assays.

  • Combine with E1, E2 enzymes, ubiquitin, ATP, and potential substrates to assess ubiquitination activity.

  • Compare wild-type CNOT4 with mutant versions to identify critical residues for activity.

• Investigation of specific known substrates:

  • RBM15 has been identified as a CNOT4 substrate that undergoes methylation-dependent ubiquitination .

  • ABCE1 is ubiquitinated by CNOT4 during mitophagy initiation .

  • Use CNOT4 antibodies in combination with antibodies against these substrates to study their interaction and modification.

• Localization studies during cellular stress:

  • CNOT4's role in the PINK1-regulated signaling pathway suggests involvement in mitochondrial quality control .

  • Use immunofluorescence with CNOT4 antibodies to track localization changes during mitochondrial damage and mitophagy.

These methodological approaches can provide insights into CNOT4's functions within the ubiquitin-proteasome system and its role in cellular processes like mitophagy and transcriptional regulation.

• What are the latest research findings regarding CNOT4's role in cancer immunotherapy and what methodological approaches should be considered for future studies?

Recent research has revealed CNOT4 as a promising factor in cancer immunotherapy, particularly for non-small cell lung cancer (NSCLC). Key findings and methodological considerations include:

These findings suggest CNOT4 could be developed as both a prognostic marker for NSCLC and a potential combinational target with anti-PD-1 treatment, opening new avenues for enhancing immunotherapy efficacy in cancer patients .

Technical and Methodological Considerations

• What are the best practices for optimizing antigen retrieval in CNOT4 immunohistochemistry?

Antigen retrieval is a critical step for successful CNOT4 immunohistochemistry. Based on the available research data, follow these methodological guidelines:

• Buffer selection:

  • Primary recommendation: TE buffer at pH 9.0 has been specifically suggested for CNOT4 antibodies .

  • Alternative approach: Citrate buffer at pH 6.0 can be used as an alternative option .

  • Compare both methods on control tissues to determine optimal conditions for your specific samples and antibody.

• Retrieval methods:

  • Heat-induced epitope retrieval (HIER): Use a pressure cooker, microwave, or water bath.

  • For pressure cooker method: Heat slides in selected buffer for 3-5 minutes at full pressure.

  • For microwave method: Heat slides in selected buffer for 10-20 minutes at medium power.

  • For water bath method: Incubate slides in selected buffer at 95-98°C for 20-30 minutes.

• Optimization strategies:

  • Titrate retrieval time: Test different durations to find the optimal balance between antigen retrieval and tissue preservation.

  • Compare different retrieval methods with the same buffer to identify the most effective approach.

  • Consider tissue-specific adjustments: Different tissue types may require modified protocols.

• Quality control measures:

  • Include positive control tissues (human lung cancer tissue has shown positive results) .

  • Process all experimental and control slides under identical conditions.

  • Document all parameters (buffer composition, pH, temperature, time) for reproducibility.

• Troubleshooting considerations:

  • Insufficient retrieval: May result in weak or absent staining.

  • Excessive retrieval: May cause tissue damage or high background.

  • If background is high: Try reducing retrieval time or temperature.

  • If signal is weak: Consider increasing retrieval time or using a different buffer system.

These methodological approaches should be optimized for each specific CNOT4 antibody and experimental system to achieve consistent and reliable immunohistochemical results.

• How should researchers approach experimental design when studying CNOT4's role in immune cell function?

When designing experiments to investigate CNOT4's role in immune cell function, particularly in the context of cancer immunotherapy, researchers should consider these methodological approaches:

• Cell and tissue models:

  • Cancer cell lines: A549 cells have been successfully used in CNOT4 studies .

  • Primary immune cells: Include T cells, particularly CD8+ cytotoxic T lymphocytes, which are increased with CNOT4 overexpression .

  • Co-culture systems: Design co-cultures of cancer cells and immune cells to study interactions.

  • In vivo models: Consider tumor-bearing mouse models with subcutaneously implanted tumor cells for studying immune infiltration .

• CNOT4 manipulation strategies:

  • Overexpression models: Generate stable CNOT4-overexpressing cell lines using lentiviral vectors .

  • Knockdown/knockout approaches: Use siRNA, shRNA, or CRISPR-Cas9 to reduce or eliminate CNOT4 expression.

  • Domain-specific mutants: Create mutants affecting specific functional domains (RING finger, RNA recognition motif) to dissect molecular mechanisms.

• Immune function assessment:

  • Flow cytometry: Quantify immune cell populations (CD3+, CD8+ T cells) and their activation status .

  • Cytokine profiling: Measure expression of lymphokines (TNF-α, IFN-γ) by qPCR or ELISA .

  • T cell functional assays: Assess cytotoxicity, proliferation, and activation of T cells in response to CNOT4 manipulation.

  • Immune cell migration: Study how CNOT4 affects immune cell recruitment to tumor sites.

• Combination therapy approaches:

  • Anti-PD-1 immunotherapy: Test in combination with CNOT4 manipulation as previously demonstrated .

  • Other checkpoint inhibitors: Explore combinations with anti-CTLA-4 or other emerging immunotherapies.

  • Targeted therapies: Investigate potential synergies with therapies targeting oncogenic pathways.

• Mechanistic investigations:

  • Ubiquitination targets: Identify immune-related proteins targeted by CNOT4's E3 ubiquitin ligase activity.

  • Transcriptional effects: Examine how CNOT4, as part of the CCR4-NOT complex, regulates immune-related gene expression.

  • Signaling pathway analysis: Investigate CNOT4's role in the JAK/STAT pathway, which it is known to activate .

• Translational considerations:

  • Patient sample analysis: Correlate CNOT4 expression with immune infiltration and treatment response in clinical samples.

  • Biomarker development: Assess CNOT4's potential as a predictive biomarker for immunotherapy response .