STK4 Antibody

What is STK4 and why is it important in research?

STK4 (also known as MST1) is a cytoplasmic kinase structurally similar to the yeast Ste20p kinase that acts upstream of stress-induced mitogen-activated protein kinase cascades. The protein can phosphorylate myelin basic protein and undergoes autophosphorylation. STK4 is particularly important in research because it plays critical roles in:

• Apoptosis regulation, where caspase-cleaved fragments of STK4 can phosphorylate histone H2B, correlating with chromatin condensation

• Immune cell function, particularly T cell survival and proliferation

• Tumor suppression, especially in contexts like hepatocellular carcinoma

STK4 deficiency is associated with autosomal recessive combined immunodeficiency (CID), making it a crucial target for immunological research .

What domains should an anti-STK4 antibody recognize for optimal detection?

When selecting an STK4 antibody, researchers should consider the functional domains of the protein and the specific research questions being addressed:

• N-terminal domain antibodies: Useful for detecting full-length STK4 protein, as demonstrated in studies of STK4 deficiency where monoclonal antibodies directed against the N-terminus successfully detected wild-type protein in controls but not in patients with certain mutations

• Kinase domain recognition: Important for studies investigating STK4's enzymatic function

• C-terminal SARAH domain: Critical for detecting dimerization capabilities, as this domain functions as a hydrophobic platform for homo- and heterodimerization

Notably, mutations truncating only the C-terminal SARAH domain can still produce detectable protein (albeit at reduced levels), whereas other mutations leading to complete protein loss require careful antibody selection for detection of any residual protein .

How do I validate STK4 antibody specificity for my experiments?

Validation of STK4 antibody specificity should include:

• siRNA knockdown controls: As demonstrated in published studies, comparing western blot results between control cells and cells transfected with STK4-specific siRNA provides strong evidence of antibody specificity

• Patient-derived cell analysis: In cases where STK4-deficient patient samples are available, these provide excellent negative controls, as seen in studies where STK4 protein was undetectable in patient T cells while intermediate levels were observed in heterozygous parents

• Protein expression correlation: Verify that detected protein levels correlate with mRNA expression levels as measured by RT-qPCR or RNA-Seq, though note that post-transcriptional mechanisms may result in discrepancies (as observed in a patient with a SARAH domain mutation who showed elevated mRNA but reduced protein levels)

• Multiple antibody comparison: Using antibodies targeting different epitopes of STK4 can confirm specificity and reveal domain-specific detection capabilities

What are the most effective methods for detecting STK4 expression in patient samples?

Based on published research, the most effective methods for detecting STK4 expression in patient samples include:

• Western blot analysis: The gold standard for quantifying STK4 protein expression, allowing detection of both full-length and truncated forms. This method effectively distinguished between complete absence of STK4 protein in some patients versus reduced levels in others

• Quantitative RT-PCR: Essential for measuring STK4 gene expression, particularly useful when protein levels are too low for reliable detection by immunoblotting

• RNA-Seq: Provides comprehensive transcriptomic analysis, allowing detection of STK4 transcript levels in context with other genes, facilitating pathway analysis

• Flow cytometry: While less commonly reported for STK4 itself, flow cytometry is valuable for assessing downstream effects of STK4 deficiency on immune cell populations and surface markers like PD-1

It's important to correlate findings across multiple detection methods, as discrepancies between mRNA and protein levels have been observed in certain STK4 mutations .

How can I assess functional impacts of STK4 deficiency or mutation using antibody-based techniques?

To assess functional impacts of STK4 deficiency or mutation, researchers can employ the following antibody-dependent approaches:

• Downstream target assessment: Immunoblotting for downstream targets like FoxO3a, which shows reduced levels in STK4-deficient cells, providing evidence of functional impairment of STK4 signaling pathways

• Phosphorylation status analysis: Using phospho-specific antibodies to detect activation states of STK4-regulated pathways, such as p65 and STAT3 phosphorylation, which are inversely associated with STK4 expression in certain contexts

• Cellular functional assays with antibody detection:

  • CFSE proliferation assays followed by flow cytometry to assess T cell proliferative capacity

  • DAPI/Annexin V staining to measure activation-induced cell death (AICD)

  • Anti-Fas antibody challenge to evaluate apoptotic responses

• Co-immunoprecipitation studies: To assess STK4 protein interactions, particularly those involving the SARAH domain that mediates homodimerization and interactions with other proteins

These techniques have been successfully employed to characterize the functional consequences of different STK4 mutations, revealing that some mutations (like those affecting only the SARAH domain) can result in milder phenotypes than complete loss-of-function mutations .

What controls should be included when using STK4 antibodies in immunoblotting?

When performing immunoblotting with STK4 antibodies, the following controls should be included:

• Positive controls:

  • Cell lines known to express STK4 (e.g., Caco-2 cells have been documented)

  • Healthy donor PBMCs or lymphoblastoid cell lines

• Negative controls:

  • siRNA knockdown samples (when possible)

  • Known STK4-deficient patient samples (if available)

• Heterozygous controls:

  • Parents of STK4-deficient patients provide excellent intermediate expression controls

• Loading controls:

  • GAPDH has been successfully used in published STK4 research

  • Other standard loading controls like β-actin are also appropriate

• Specificity controls:

  • Competing peptide assays

  • Multiple antibodies targeting different epitopes of STK4

Research has shown that proper controls are critical for interpreting STK4 expression data, particularly in cases where mutations result in truncated but partially functional protein rather than complete absence .

How can STK4 antibodies be used to investigate the relationship between STK4 and TLR signaling pathways?

STK4 antibodies can be instrumental in exploring the interplay between STK4 and Toll-like receptor (TLR) signaling:

• Co-immunoprecipitation studies: Using STK4 antibodies to pull down protein complexes can reveal interactions with TLR pathway components. Research has identified relationships between STK4 and TLR pathway mediators, particularly IRAK1

• Comparative expression analysis: Immunoblotting for STK4 alongside TLR pathway components (like IRAK1) in paired samples can reveal inverse relationships, as observed in macrophages isolated from hepatocellular carcinoma patients where STK4 expression was inversely associated with IRAK1, IL-6, and phosphorylated p65 or STAT3

• Pathway intervention studies: Combining STK4 antibody detection with TLR pathway inhibitors (such as IRAK1/4 inhibitors) can help determine causality in observed correlations. In mouse models, IRAK1/4 inhibitor treatment affected IL-6 levels and tumor development in STK4-deficient contexts

• Stimulus-response experiments: Using STK4 antibodies to track protein levels and modifications following TLR stimulation (e.g., with LPS or bacterial components) can elucidate dynamic relationships between these pathways

These approaches can provide insights into how STK4 regulates inflammatory responses through TLR pathways, with implications for both infectious disease and cancer research.

What are the challenges in detecting truncated or mutant forms of STK4 protein?

Detecting truncated or mutant forms of STK4 presents several technical challenges that researchers should consider:

• Epitope availability: Depending on the mutation, epitopes recognized by particular antibodies may be absent or altered. For example, C-terminal mutations may not affect detection by N-terminal-targeting antibodies, as seen in a patient with a mutation truncating only the SARAH domain where the protein was still detectable, albeit at reduced levels

• Protein stability differences: Mutations can affect protein stability, leading to lower steady-state levels despite normal or even elevated mRNA expression. This was observed in a case where STK4 mRNA was significantly higher than in controls, but the protein level was significantly lower due to a C-terminal truncation affecting the SARAH domain

• Dimerization-dependent detection: Since wild-type STK4 predominantly exists as homodimers, mutations affecting dimerization (particularly in the SARAH domain) may alter antibody accessibility or protein conformation

• Nuclear vs. cytoplasmic localization: STK4 can shuttle between cytoplasmic and nuclear compartments depending on its activation state. Mutations affecting autophosphorylation or nuclear localization signals may alter this distribution, requiring subcellular fractionation for accurate detection

• Cross-reactivity with paralogs: STK4 has an important paralog, STK3, which could potentially cross-react with some antibodies, requiring careful specificity testing

To address these challenges, researchers should consider using multiple antibodies targeting different regions of the protein and correlating protein detection with mRNA expression data.

How can STK4 antibodies help differentiate between clinical phenotypes of STK4 deficiency?

STK4 antibodies play a crucial role in differentiating between clinical phenotypes of STK4 deficiency by enabling detailed molecular characterization:

• Residual protein detection: Different mutations in STK4 can lead to varying levels of protein expression, from complete absence to reduced levels of full-length or truncated protein. Western blotting with appropriate antibodies can quantify these differences, which have been correlated with clinical severity

• Functional domain analysis: Using domain-specific antibodies can reveal whether particular functional domains remain intact. For instance, a patient with a mutation affecting only the SARAH domain exhibited a milder clinical phenotype despite severe T cell lymphopenia, contrasting with more severe presentations in patients with complete protein loss

• Downstream pathway assessment: Immunoblotting for STK4-regulated proteins (like FoxO3a) provides insight into the functional consequences of different mutations. The degree of dysregulation in these pathways correlates with clinical manifestations

This molecular stratification using antibody-based techniques has important implications for treatment decisions, as patients with milder molecular phenotypes may require less aggressive interventions than those with complete STK4 deficiency .

How can STK4 antibodies contribute to understanding the role of STK4 in tumor suppression?

STK4 antibodies are valuable tools for investigating STK4's tumor suppressor functions, particularly in liver cancer:

• Expression correlation studies: Immunoblotting for STK4 in paired tumor and non-tumor tissues can reveal expression patterns associated with cancer progression. Research has shown that STK4 expression is markedly reduced in macrophages isolated from human hepatocellular carcinoma (HCC) patients

• Pathway interaction analysis: Using STK4 antibodies alongside detection of inflammatory mediators (IL-6, phospho-STAT3, phospho-p65) can elucidate mechanisms by which STK4 regulates tumor-promoting inflammation. Studies have demonstrated inverse correlations between STK4 and these inflammatory markers in HCC patients

• Serum biomarker evaluation: While direct antibody applications typically involve tissue or cellular analysis, research has shown that serum STK4 levels were specifically decreased in HCC patients with high levels of IL-6, suggesting potential for circulating STK4 as a diagnostic biomarker

• Therapeutic intervention assessment: STK4 antibodies can help evaluate the effectiveness of targeted interventions in STK4-deficient contexts. For example, in STK4-deficient mice, treatment with an IRAK1/4 inhibitor reduced serum IL-6 levels and liver tumor numbers, suggesting mechanistic links between STK4 deficiency, inflammation, and tumorigenesis

These applications highlight STK4's potential dual role in both immune regulation and tumor suppression, with antibody-based detection methods providing crucial insights for translational research.

What methodological approaches can resolve discrepancies between STK4 mRNA and protein levels?

Researchers facing discrepancies between STK4 mRNA and protein levels should consider these methodological approaches:

• Comprehensive protein stability assessment:

  • Pulse-chase experiments with protein synthesis inhibitors to determine protein half-life

  • Proteasome inhibitors (e.g., MG132) to assess involvement of proteasomal degradation

  • Analysis of post-translational modifications affecting stability

• Translation efficiency evaluation:

  • Polysome profiling to assess mRNA translation status

  • Analysis of translation initiation factors interacting with STK4 mRNA

• Targeted mutation impact studies:

  • For known mutations (like those affecting the SARAH domain), generate construct variants to directly test effects on protein stability

  • Complementation experiments in deficient cells to verify functional consequences

• Structural biology approaches:

  • In silico modeling of how mutations affect protein folding and stability

  • Comparison with known effects of mutations in similar protein domains

These approaches are particularly relevant given published observations of a patient with a SARAH domain truncation mutation who exhibited significantly higher STK4 mRNA levels than controls but significantly lower protein levels, suggesting that the inability to homodimerize may have reduced protein stability .

How can STK4 antibodies help characterize the intersection of STK4 signaling with immune response pathways?

STK4 antibodies provide powerful tools for exploring the complex interactions between STK4 signaling and immune response pathways:

• Stimulus-response profiling: Using STK4 antibodies to track protein levels and modifications following immune stimulation can reveal regulatory dynamics. Studies have employed RNA-Seq and protein analysis of PBMCs stimulated with IFN-α/IFN-β or PMA/ionomycin to identify STK4-dependent gene expression patterns

• Regulatory network mapping:

  • Combining STK4 immunoprecipitation with mass spectrometry can identify novel interaction partners in immune contexts

  • Chromatin immunoprecipitation (ChIP) with STK4 antibodies can reveal direct genomic targets

• Single-cell analysis: Using STK4 antibodies in flow cytometry or mass cytometry (CyTOF) protocols allows correlation of STK4 expression with cellular phenotypes at single-cell resolution

• Phospho-protein network analysis: Multiplex phospho-protein detection alongside STK4 can map signaling networks. Research has identified regulatory networks of IFN-α/IFN-β-responsive genes encompassing cytokine, chemokine, and adhesion factor genes that are indirectly regulated by STK4

These approaches have revealed that STK4 deficiency leads to marked dysregulation, but not complete abrogation, of IFN-regulated gene expression, highlighting the nuanced role of STK4 in immune signaling networks .

What are the common technical challenges when using STK4 antibodies and how can they be addressed?

Researchers working with STK4 antibodies may encounter several technical challenges:

• Low signal intensity: STK4 protein levels may be naturally low or reduced in certain conditions

  • Solution: Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • Solution: Optimize protein extraction methods to ensure complete recovery

  • Solution: Increase antibody incubation time or concentration, following titration experiments

• High background in Western blots:

  • Solution: Increase blocking time or concentration

  • Solution: Try alternative blocking agents (milk vs. BSA)

  • Solution: Optimize antibody dilution through systematic titration

  • Solution: Include additional washing steps with higher detergent concentration

• Cross-reactivity with paralogs:

  • Solution: Verify antibody specificity using siRNA knockdown controls

  • Solution: Consider using monoclonal antibodies with validated epitope specificity

  • Solution: Include known STK4-deficient samples as negative controls when possible

• Inconsistent results in patient samples:

  • Solution: Standardize sample collection and processing protocols

  • Solution: Consider clinical factors that might influence STK4 expression

  • Solution: Always include appropriate controls (healthy, heterozygous, and known deficient where available)

• Detection of truncated proteins:

  • Solution: Use antibodies targeting different epitopes

  • Solution: Optimize gel percentage and running conditions for lower molecular weight proteins

  • Solution: Consider using gradient gels to resolve proteins of varying sizes

These solutions are based on successful approaches documented in STK4 research, where careful optimization of detection methods revealed important biological insights about protein expression in various mutant contexts .

How should STK4 antibody-based assays be adapted for different cell types and tissues?

Optimizing STK4 antibody-based assays for different biological contexts requires specific adaptations:

• Immune cells (PBMCs, T cells):

  • Protein extraction: Use gentle lysis buffers to preserve phosphorylation status

  • Detection strategy: Consider flow cytometry for rare populations

  • Stimulation protocols: Include relevant T cell activators (CD3/CD28) when assessing functional impacts

• Liver tissues and hepatocytes:

  • Sample preparation: Use specialized extraction buffers to overcome lipid interference

  • Background reduction: Additional washing steps may be needed

  • Context considerations: Assess STK4 alongside inflammation markers (IL-6, phospho-STAT3)

• Cell lines vs. primary cells:

  • Antibody titration: Primary cells often require different antibody concentrations than cell lines

  • Signal amplification: Primary cells may need more sensitive detection methods

  • Controls: Include relevant cell-type specific positive and negative controls

• Tissue-specific considerations:

  • Fixation protocols: Optimize fixation methods to preserve epitope accessibility

  • Antigen retrieval: Different tissues may require specific retrieval methods

  • Autofluorescence management: Use appropriate quenching methods for immunofluorescence applications

These adaptations should be empirically determined for each experimental context, with careful documentation of optimization steps to ensure reproducibility across studies.

What are the best practices for quantifying STK4 protein levels in comparative studies?

For accurate quantification of STK4 protein in comparative studies, researchers should follow these best practices:

• Standardized loading controls:

  • Use consistent loading controls across all samples (GAPDH has been successfully used in STK4 research)

  • Verify linear range of loading control detection to ensure accurate normalization

  • Consider multiple loading controls when comparing diverse tissue types

• Quantification methodology:

  • Use digital image analysis software (e.g., ImageJ has been used successfully for STK4 quantification)

  • Ensure images are captured within the linear dynamic range of the detection system

  • Perform replicate experiments (at least three independent immunoblots for statistical validity)

• Statistical analysis:

  • Apply appropriate statistical tests (e.g., unpaired Student's t-test has been used to compare STK4 expression between patient and control samples)

  • Report both mean values and measures of variation (standard error of the mean or standard deviation)

  • Consider power calculations to determine appropriate sample sizes

• Reporting standards:

  • Present both raw blot images and quantification graphs

  • Clearly state normalization methods

  • Report all relevant experimental details (antibody source, dilution, incubation conditions)

• Validation with orthogonal methods:

  • Correlate protein levels with mRNA expression when possible

  • Consider using ELISA or other quantitative protein detection methods as complementary approaches

These practices have been implemented in published STK4 research, resulting in rigorous quantitative comparisons that revealed significant differences in protein expression between patient and control samples, even in cases with subtle phenotypic variations .