MAP4K4 Antibody

What is MAP4K4 and why is it significant in research?

MAP4K4, also known as NIK, HGK, and several other aliases, belongs to the protein kinase superfamily, specifically the STE Ser/Thr protein kinase family and STE20 subfamily. It functions as a serine/threonine kinase that responds to environmental stress and cytokines such as TNF-alpha . MAP4K4 is significant in research because it operates upstream of the JUN N-terminal pathway and acts as an activator of the Hippo signaling pathway, which plays a pivotal role in organ size control and tumor suppression . Its overexpression in various cancers makes it an important target for oncology research and potential therapeutic development .

What applications are MAP4K4 antibodies validated for?

MAP4K4 antibodies have been validated for multiple experimental applications. The most common applications include:

It is recommended to optimize dilutions for each specific testing system to obtain optimal results .

What are the recommended storage conditions for MAP4K4 antibodies?

MAP4K4 antibodies should be stored at -20°C where they remain stable for one year after shipment. For specific preparations, such as those in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, aliquoting is generally unnecessary for -20°C storage. Some preparations (20μl sizes) may contain 0.1% BSA . Always check the manufacturer's instructions for specific storage recommendations as formulations may vary between suppliers.

How should I design experiments to validate MAP4K4 knockdown efficiency?

When designing experiments to validate MAP4K4 knockdown, a multi-level validation approach is recommended:

• Vector Construction: Design at least two shRNA sequences targeting different regions of the MAP4K4 gene along with a control shRNA sequence. These can be constructed into lentiviral vectors such as pLVX-shRNA2-Puro .

• Transfection and Selection: After transfection into packaging cells (e.g., 293T cells), collect virus-containing supernatant and filter through a 0.45μm filter. Infect target cells and select with puromycin (typically 3μg/mL) to establish stable cell lines .

• Validation at mRNA Level: Extract RNA and perform quantitative RT-PCR to confirm reduced MAP4K4 mRNA expression.

• Validation at Protein Level: Perform Western blot analysis to confirm reduced MAP4K4 protein expression.

• Functional Validation: Assess cellular phenotypes such as proliferation, cell cycle progression, or pathway activation that are known to be affected by MAP4K4.

Select the shRNA sequence demonstrating the best knockdown efficiency for subsequent experimental research .

What controls should be included when using MAP4K4 antibodies in cancer research?

When conducting cancer research with MAP4K4 antibodies, include the following controls:

• Positive Controls: Use cell lines known to express MAP4K4, such as Jurkat or PC-3 cells for Western blotting .

• Negative Controls:

  • Primary antibody omission controls

  • Isotype controls (rabbit IgG for rabbit polyclonal antibodies)

  • Ideally, MAP4K4 knockdown or knockout samples when available

• Tissue Controls: For IHC, include human skeletal muscle, placenta, or liver tissue as positive controls .

• Adjacent Normal Tissue Controls: When examining tumor samples, always include paired adjacent non-tumor tissue to establish baseline expression and evaluate overexpression .

• Loading Controls: For Western blots, include appropriate housekeeping proteins (β-actin, GAPDH, etc.) to normalize protein loading.

• Antibody Specificity Controls: When possible, verify specificity with peptide blocking experiments or multiple antibodies targeting different epitopes.

What is the optimal antigen retrieval method for MAP4K4 immunohistochemistry?

For optimal MAP4K4 detection in immunohistochemistry, the recommended antigen retrieval method is:

Primary method: TE buffer at pH 9.0

Alternative method: Citrate buffer at pH 6.0

The choice between these methods may depend on tissue type and fixation conditions. For human skeletal muscle, placenta, and liver tissues, both methods have been validated, with TE buffer pH 9.0 generally yielding better results. It is advisable to test both retrieval methods on your specific tissue samples to determine which provides optimal staining with minimal background .

How can I troubleshoot non-specific binding in Western blot when using MAP4K4 antibodies?

When encountering non-specific binding in Western blot with MAP4K4 antibodies, implement these troubleshooting steps:

• Confirm Molecular Weight: MAP4K4 has a calculated molecular weight of 151 kDa but is typically observed between 130-160 kDa on gels . Ensure your bands fall within this range.

• Optimize Blocking: Increase blocking time or test different blocking agents (5% BSA may be more effective than milk for phosphoprotein detection).

• Dilution Optimization: Titrate antibody concentration, starting with 1:500 dilution and adjusting based on signal-to-noise ratio .

• Increase Washing: Extend washing steps with TBS-T to reduce background.

• Add Detergents: Include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific binding.

• Pre-adsorption: If available, pre-adsorb antibody with recombinant MAP4K4 protein to test specificity.

• Use Fresh Antibody: Avoid repeated freeze-thaw cycles which can increase non-specific binding.

• Validate with KD/KO Controls: Utilize MAP4K4 knockdown or knockout samples as negative controls to confirm band specificity .

How can MAP4K4 antibodies be used to investigate its role in cancer radioresistance?

MAP4K4 antibodies are crucial tools for investigating radioresistance mechanisms, as demonstrated in recent breast cancer research . A comprehensive approach includes:

• Establish Radioresistant Models: Generate radioresistant cell lines by exposing parental cells to fractionated radiation (e.g., 4-5 Gy twice weekly for several months) .

• Validate MAP4K4 Expression Changes:

  • Use Western blot with MAP4K4 antibodies to quantify protein levels in parental vs. radioresistant cells

  • Confirm with immunofluorescence to assess subcellular localization changes

• Pathway Analysis: Combine MAP4K4 antibodies with antibodies against downstream targets to map signaling cascade changes:

  • DNA damage response proteins (γH2AX, 53BP1)

  • Apoptosis markers (cleaved caspase-3, PARP)

  • ACSL4, which has been identified as a downstream target of MAP4K4 in radioresistance

• Inhibitor Studies: Use MAP4K4 inhibitors (PF06260933, GNE-495) in combination with radiation and assess:

  • Changes in MAP4K4 phosphorylation status

  • Effects on DNA damage accumulation

  • Apoptotic response

  • Clonogenic survival

• Mechanistic Validation: Perform MAP4K4 knockdown/overexpression experiments and use the antibody to confirm expression changes and correlate with radioresistance phenotypes .

This approach has revealed that MAP4K4 plays a crucial role in radioresistance by acting upstream of ACSL4 to enhance DNA damage response and inhibit apoptosis in breast cancer cells .

What are the considerations for using MAP4K4 antibodies in mapping tumor immune microenvironments?

When using MAP4K4 antibodies to study tumor immune microenvironments, consider these research-driven approaches:

• Multiplex Immunohistochemistry/Immunofluorescence:

  • Combine MAP4K4 antibodies with immune cell markers (CD3, CD4, CD8, CD68, etc.)

  • Use spectral unmixing to differentiate signals

  • Analyze spatial relationships between MAP4K4-expressing cells and immune infiltrates

• Correlation with Immune Signatures:

  • Use bioinformatic tools like ESTIMATE to evaluate stromal and immune cell infiltration scores

  • Apply CIBERSORT to deconvolute expression matrices of 22 immune cell subtypes

  • Correlate MAP4K4 expression levels with specific immune cell populations

• Functional Validation:

  • In co-culture experiments, assess how MAP4K4 inhibition or overexpression affects immune cell activation

  • Measure cytokine production in MAP4K4-manipulated cancer cells using antibody-based assays

• Patient Sample Analysis:

  • Stratify patient samples into high and low MAP4K4 expression groups

  • Compare immune infiltration patterns between groups

  • Correlate with clinical outcomes and treatment response

Research has shown that MAP4K4 expression levels correlate with specific immune scores in gastric cancer, suggesting its role in modulating the tumor immune microenvironment .

How can I integrate MAP4K4 antibody-based techniques with genomic and transcriptomic data?

To effectively integrate MAP4K4 antibody-based techniques with genomic and transcriptomic approaches:

• Multi-omics Correlation:

  • Compare protein expression levels detected by MAP4K4 antibodies with mRNA expression from RNA-seq

  • Investigate potential post-transcriptional regulation mechanisms when discrepancies exist

  • Correlate with gene copy number variations or mutations from genomic data

• Pathway Analysis:

  • Use MAP4K4 antibodies to validate key nodes identified in pathway enrichment analyses

  • In gastric cancer research, MAP4K4 high-expression groups showed enrichment in tumor-related pathways such as PI3K-Akt signaling

• Clinical Stratification:

  • Stratify patients based on MAP4K4 protein expression (by IHC)

  • Compare with transcriptomic clusters

  • Correlate with survival outcomes and treatment responses

• Regulatory Network Analysis:

  • Use MAP4K4 antibodies to validate predicted miRNA and transcription factor regulatory relationships

  • Cross-reference with miRDB, mirtarbase, miRMap, miRanda, and TargetScan databases

  • Confirm protein-level changes following manipulation of predicted regulators

• Functional Validation:

  • Validate transcriptomic findings using targeted protein approaches

  • For genes identified in differential expression analyses, confirm protein-level changes using appropriate antibodies alongside MAP4K4

This integrated approach has proven valuable in hepatocellular carcinoma research, where MAP4K4 overexpression was confirmed at both protein and mRNA levels and associated with clinical outcomes .

What are the best practices for combining MAP4K4 antibodies with patient-derived xenograft (PDX) models?

When incorporating MAP4K4 antibodies in PDX model research, follow these research-driven best practices:

• Baseline Characterization:

  • Use MAP4K4 antibodies to assess expression in original patient tumor samples

  • Compare expression levels between original tumors and established PDX models across passages

  • Document any expression changes during PDX establishment

• Experimental Design:

  • Stratify PDX models based on MAP4K4 expression levels

  • Design treatment groups considering MAP4K4 status

  • Include appropriate controls for antibody validation within PDX tissues

• Treatment Response Monitoring:

  • Use MAP4K4 antibodies to monitor expression changes during treatment

  • Perform IHC on sequential biopsies or terminal samples

  • Correlate changes with treatment outcomes

• Combination with Functional Assays:

  • When testing MAP4K4 inhibitors, use antibodies to confirm target engagement

  • Assess downstream pathway modulation via phospho-specific antibodies

  • Correlate with phenotypic outcomes (tumor growth, metastasis)

• Species-Specific Considerations:

  • Select MAP4K4 antibodies that can distinguish between human (tumor) and mouse (stroma) proteins

  • Consider dual immunofluorescence with species-specific markers

  • When analyzing whole tumor lysates, account for potential stromal contribution

These approaches help establish MAP4K4 as a therapeutic target and potential biomarker in personalized oncology applications.

How are MAP4K4 antibodies being used to explore its role in tumor radioresistance mechanisms?

Recent research has revealed MAP4K4 as a critical mediator of radioresistance, particularly in breast cancer. MAP4K4 antibodies are instrumental in exploring these mechanisms through:

• Expression Correlation Studies:

  • MAP4K4 antibodies have revealed significantly elevated expression in radioresistant breast cancer cells compared to parental lines, regardless of breast cancer subtype

  • Western blot analysis using MAP4K4 antibodies helps quantify these expression differences

• Mechanism Exploration:

  • Antibodies against MAP4K4 and its downstream targets help elucidate the molecular pathway of radioresistance

  • Research has identified that MAP4K4 functions upstream of ACSL4 in radioresistant breast cancer cells

  • Antibody-based studies have shown MAP4K4 inhibition suppresses ACSL4 expression

• Therapeutic Target Validation:

  • MAP4K4 antibodies confirm target engagement of MAP4K4 inhibitors like PF06260933 and GNE-495

  • They help visualize the accumulation of DNA damage through γH2AX staining following MAP4K4 inhibition

  • They verify the inhibition of DNA repair systems in radioresistant cells treated with MAP4K4 inhibitors

• Patient Stratification Research:

  • IHC with MAP4K4 antibodies helps stratify patient samples based on expression levels

  • This stratification can potentially identify patients who might benefit from MAP4K4 inhibitors as radiosensitizers

These applications of MAP4K4 antibodies have contributed to the groundbreaking finding that MAP4K4 inhibition can overcome radioresistance in breast cancer cells by enhancing DNA damage and promoting apoptosis .

What are the methodological considerations for studying MAP4K4 in multi-cancer type comparisons?

When using MAP4K4 antibodies for cross-cancer comparisons, consider these methodological approaches:

• Standardized Protocols:

  • Maintain consistent antibody dilutions across cancer types (recommended 1:200-1:1000 for WB, 1:20-1:200 for IHC)

  • Use identical antigen retrieval methods (preferably TE buffer pH 9.0)

  • Standardize detection systems to ensure comparable sensitivity

• Tissue Microarray (TMA) Applications:

  • Design TMAs containing multiple cancer types with matched normal tissues

  • Include internal control tissues known to express MAP4K4 (skeletal muscle, placenta, liver)

  • Use automated scoring systems to reduce subjective interpretation

• Cancer-Specific Validation:

  • Validate MAP4K4 antibody performance in each cancer type independently

  • Establish positive controls specific to each cancer type

  • Document cancer-specific molecular weight variations or isoform expression

• Integrated Analysis Approach:

  • Combine IHC findings with cancer-specific transcriptomic data

  • Correlate MAP4K4 protein expression with cancer-specific driver mutations

  • Analyze MAP4K4 expression in relation to cancer-specific microenvironment factors

• Functional Context:

  • Consider cancer-specific pathway interactions (MAP4K4 regulates different downstream targets in different cancers)

  • In hepatocellular carcinoma, focus on JNK, NFκB, and toll-like receptor pathways

  • In gastric cancer, examine PI3K-Akt pathway connections

  • In breast cancer, investigate DNA damage response and ACSL4 relationships

This methodological framework enables meaningful cross-cancer comparisons while respecting cancer-specific biological contexts.