STK4 Antibody, FITC conjugated

What is STK4 and why is it important in immunological research?

STK4 (Serine/Threonine Kinase 4, also known as MST1) is a critical regulatory protein involved in multiple biological processes including:

• Regulation of cell proliferation, differentiation, and apoptosis in immune cells

• T cell immunity through TCR-responsive regulation of transcription

• Formation of a trimolecular complex with Foxp3 and NF-κB p65 to promote Treg cell function

• Lymphocyte adhesion and trafficking through interaction with the RapL/Rap1 module

STK4 deficiency causes a primary immunodeficiency syndrome characterized by T and B cell lymphopenia, intermittent neutropenia, and atrial septal defects. Patients with STK4 deficiency typically present with recurrent bacterial and viral infections, mucocutaneous candidiasis, cutaneous warts, and skin abscesses . The protein is broadly expressed in hematopoietic cells, particularly monocytes, and is not restricted to lymphocytes .

What are the recommended applications for STK4 FITC-conjugated antibodies?

STK4 FITC-conjugated antibodies are versatile tools with multiple research applications:

Flow cytometry has been validated as a rapid and reliable method to detect STK4 protein expression and is qualified as a diagnostic tool to study STK4 deficiency. This approach allows for calculation of ΔMFI/cell values to investigate differences in protein expression among subjects .

How should researchers preserve FITC-conjugated antibody fluorescence intensity?

FITC (Fluorescein Isothiocyanate) conjugated antibodies require special handling to maintain optimal performance:

• Store at -20°C in the dark with included stabilizers (commonly PBS, 0.02% NaN3, BSA, and 50% glycerol)

• Do not expose to continuous light as this will cause the FITC-conjugated antibody to gradually lose its fluorescence

• Avoid repeated freeze-thaw cycles

• For working solutions, store at 2-8°C for up to 6 months after reconstitution

• Centrifuge briefly before opening to ensure collection of the entire product

• Use amber tubes or aluminum foil wrapping during experimental procedures

For long-term storage, aliquoting the antibody into smaller volumes is recommended to minimize freeze-thaw cycles and preserve activity.

What controls are essential when using STK4 FITC-conjugated antibodies?

When conducting experiments with STK4 FITC-conjugated antibodies, the following controls are crucial:

Positive Controls:

• Cell lines with known STK4 expression (HeLa cells show both nuclear and cytoplasmic expression)

• Human PBMCs from healthy donors (STK4 is highly expressed)

• Purified Positope™ control protein for immunoblotting verification

Negative Controls:

• PBMCs from STK4-deficient patients (for clinical research)

• Isotype control antibodies of matching IgG subclass (IgG1, IgG2a, or IgG2b)

• Secondary antibody-only controls for indirect immunofluorescence

Validation Controls:

• Heterozygous STK4 mutation carriers (show intermediate expression levels)

• Blocking peptide competition assays

• siRNA knockdown samples

These controls help establish specificity and minimize the risk of false positive or negative results in experimental protocols.

How can STK4 FITC-conjugated antibodies be optimized for flow cytometry in diagnostic applications?

Flow cytometry has been validated as a reliable method for STK4 protein detection with important optimization considerations:

Protocol Optimization:

• Isolate PBMCs using density gradient centrifugation

• Fix cells with 4% paraformaldehyde and permeabilize with appropriate detergent

• Test different permeabilization reagents (0.1-1% Triton X-100 recommended)

• Optimize antibody concentration (typically 1:50-1:200 dilution) to maximize signal-to-noise ratio

• Include established protocols for intracellular staining of other relevant markers

Analysis Parameters:

• Calculate ΔMFI/cell values to normalize STK4 expression across samples

• Establish threshold values based on healthy control samples

• Validate results with Western blotting in parallel

• Combine with markers for specific lymphocyte subpopulations

In STK4 deficiency diagnosis, flow cytometry data shows reduced STK4 protein expression in the peripheral blood mononuclear cells obtained from all STK4-deficient patients compared to healthy controls . This approach allows for rapid screening of suspected cases before genetic confirmation.

What methodological approaches are recommended for studying STK4 subcellular localization upon T cell activation?

STK4 exhibits dynamic subcellular localization that is critical to its function:

Experimental Design:

• Stimulate T cells with anti-CD3/CD28 antibodies to activate TCR signaling

• Collect cells at multiple timepoints (0, 0.5h, 1h, 2h, 4h) to capture translocation dynamics

• Fix with 4% paraformaldehyde and permeabilize with 1% Triton X-100

• Stain with STK4 FITC-conjugated antibody and nuclear markers

Advanced Analysis:

• Use confocal microscopy for high-resolution localization

• Perform co-localization studies with Foxp3 and NF-κB p65

• Include the STK4 kinase inhibitor XMU-MP-1 to demonstrate kinase-dependent translocation

• Compare wild-type STK4 with kinase-dead (K59R) or NLS mutant variants

Research shows that STK4 is predominantly localized to the cytosol in quiescent Treg cells, and stimulation with anti-CD3/CD28 mAbs induces time-dependent translocation into the nucleus as early as 0.5h post-stimulation, where it co-localizes with Foxp3 . This translocation is inhibited by treatment with XMU-MP-1, indicating a kinase activity-dependent mechanism.

How should researchers approach STK4 detection in samples from patients with immune deficiencies?

Detecting STK4 in clinical samples requires specialized approaches:

Multi-modal Assessment:

• Flow Cytometry Screening:

  • Measure STK4 expression in different immune cell populations

  • Compare with age-matched controls

  • Calculate ΔMFI values to standardize results across laboratories

• Functional Testing:

  • Assess T cell activation and apoptosis susceptibility

  • Evaluate interferon signaling responses

  • Test neutrophil function and survival

• Genetic Confirmation:

  • Whole genome or targeted sequencing

  • Sanger sequencing of candidate mutations

  • Analysis of STK4 mRNA expression

When investigating suspected STK4 deficiency, researchers should note that heterozygous carriers display intermediate levels of STK4 protein expression, while homozygous patients show absence or severe reduction of the protein . Complementary approaches include measuring type I/II and III interferon responses to various TLR agonists, as STK4-deficient cells show impaired signaling .

What are the recommended methods for investigating STK4's role in the formation of protein complexes?

To study STK4's interactions with binding partners:

Co-immunoprecipitation Strategy:

• Stimulate cells with appropriate activators (anti-CD3/CD28 for T cells)

• Prepare lysates with buffers that preserve protein-protein interactions

• Immunoprecipitate with anti-STK4 antibody

• Analyze precipitated complexes for Foxp3 and p65

• Perform reverse IPs with anti-Foxp3 or anti-p65 antibodies

Validation Approaches:

• Use HEK293T overexpression systems to confirm direct interactions

• Test kinase-dead STK4 mutants (K59R) to evaluate kinase-dependency

• Examine the effect of STK4 phosphorylation on complex formation

• Study the formation of the trimolecular STK4-Foxp3-p65 complex using sequential IPs

Research has demonstrated that TCR stimulation induces the association of Foxp3 with p65 and also promotes the formation of a larger trimolecular complex including STK4, Foxp3, and p65 . This complex appears to be stabilized by STK4-mediated phosphorylation of Foxp3 on Serine 418, highlighting the importance of STK4's enzymatic activity.

How can researchers evaluate STK4 expression patterns in different disease contexts?

To systematically analyze STK4 expression in disease states:

Tissue Sample Analysis:

• Perform immunohistochemistry with standardized scoring systems

• Analyze STK4 expression on a scale: negative (0), low (+/1), medium (++/3), strong (+++/5)

• Use statistical methods like the Wilcoxon matched-pairs test to calculate significance of differences

• Correlate expression with clinical parameters (tumor grade, disease stage)

Database Integration:

• Utilize databases like Oncomine, GTEX Portal, and CCLE for comparative analysis

• Retrieve survival data from the Human Protein Atlas

• Perform Kruskal-Wallis test for non-parametric values to correlate with clinical parameters

• Use GraphPad Prism software for multiple comparisons of nonparametric criteria

For cancer studies, researchers should note that STK4 has been identified as a tumor suppressor in hepatocellular carcinoma, breast cancer, and lymphoma through regulation of cell differentiation and apoptosis . Expression patterns may vary significantly between tumor and normal tissues, offering potential prognostic value.

What technical considerations are important when studying STK4 in neutrophil populations?

Studying STK4 in neutrophils presents unique challenges:

Isolation and Preservation:

• Use density gradient techniques optimized for neutrophil isolation

• Process samples immediately to minimize spontaneous apoptosis

• Include protease inhibitors in all buffers

Functional Assessments:

• Measure apoptosis rates using Annexin V/PI staining

• Evaluate mitochondrial membrane potential

• Assess neutrophil chemotaxis and phagocytic capacity

Species-Specific Considerations:

• Note that neutrophil STK4 expression differs between humans and mice

• Human neutrophils express detectable STK4 despite earlier negative reports

• Species-dependent mechanisms controlling neutrophil viability have been noted in comparative studies

STK4-deficient neutrophils exhibit enhanced loss of mitochondrial membrane potential and increased susceptibility to apoptosis . Intermittent neutropenia observed in STK4-deficient patients may result from increased apoptosis rather than defective production, as bone marrow samples show no evidence of a block in myeloid differentiation .

What approaches are recommended for studying the role of STK4 in interferon signaling pathways?

To investigate STK4's role in interferon responses:

Stimulation Protocols:

• Stimulate cells with type I IFNs (IFNα/β) at standardized concentrations

• Include TLR agonists (LPS, poly(I:C)) to assess TLR3/4 pathways

• Use live viral challenges where appropriate

• Design time course experiments (2h, 4h, 8h, 24h)

Readout Systems:

• Measure phosphorylation of TBK1 and IRF3 by immunoblotting

• Analyze IFN-responsive gene expression by RT-qPCR or RNA-Seq

• Perform gene enrichment analyses on subsets of IFN-α/IFN-β-responsive genes

• Use ClueGO and Cytoscape for functional grouped GO and pathway annotation networks

STK4 deficiency leads to dysregulated interferon signaling, particularly defective type I/II and III interferon responses to various TLR agonists, live viruses, and bacterial lysates. This appears to be due to impaired phosphorylation of the kinase TBK1 and the transcription factor IRF3 , which are critical components of the interferon production pathway.

How can researchers differentiate between the pro-apoptotic and anti-apoptotic functions of STK4?

STK4 exhibits context-dependent roles in cellular survival:

Experimental Approaches:

• For Pro-apoptotic Functions:

  • Monitor STK4 cleavage by caspases (63-kDa → 36-kDa fragment)

  • Track nuclear translocation of the N-terminal fragment

  • Assess histone phosphorylation

  • Examine JNK pathway activation

• For Anti-apoptotic Functions:

  • Measure mitochondrial membrane potential in STK4-deficient vs. normal cells

  • Assess Annexin V staining and caspase activation

  • Evaluate FOXO signaling pathway components

  • Monitor cell survival over extended culture periods

• Distinguishing Methods:

  • Use cell-type specific assays (T cells vs. neutrophils vs. monocytes)

  • Compare results in resting vs. activated states

  • Include both early (4-6h) and late (24-48h) timepoints

What are the latest methodological approaches for monitoring STK4 phosphorylation status?

Recent advances in studying STK4 phosphorylation include:

Antibody-Based Methods:

• Phospho-specific antibodies targeting known STK4 phosphorylation sites

• Phos-tag™ SDS-PAGE for mobility shift detection

• Proximity ligation assays for detecting phosphorylated species in situ

Mass Spectrometry Approaches:

• Targeted phosphoproteomics focusing on STK4 and its substrates

• SILAC labeling for quantitative assessment of phosphorylation dynamics

• Parallel reaction monitoring for absolute quantification

Functional Readouts:

• Monitoring phosphorylation of known substrates (e.g., MOB1)

• Assessing kinase activity using non-radioactive ATP consumption assays

• Develop reporter constructs with phosphorylation-dependent localization changes

Research has shown that STK4 phosphorylates Foxp3 on Serine 418, which stabilizes the STK4-Foxp3-p65 complex formation . Similarly, STK4-mediated phosphorylation of other substrates like MOB1 is critical for downstream signaling events . These phosphorylation events represent key regulatory mechanisms that can be monitored to assess STK4 function.

How can multiparameter analysis be optimized when using STK4 FITC-conjugated antibodies?

For complex multiparameter studies:

Panel Design Considerations:

• Place STK4-FITC appropriately within the panel based on its expression level

• Consider spectral overlap with other fluorochromes (PE, APC, PerCP)

• Use compensation controls for each fluorochrome

• Include viability dyes compatible with fixed/permeabilized cells

Optimization Strategy:

• Titrate STK4 FITC-conjugated antibody using positive control samples

• Test different fixation/permeabilization protocols to optimize for all markers

• Confirm antibody performance in multiplex conditions

• Include Fluorescence Minus One (FMO) controls for accurate gating

Data Analysis Approaches:

• Apply dimensionality reduction techniques (tSNE, UMAP)

• Perform unsupervised clustering to identify cell populations

• Correlate STK4 expression with other functional markers

• Normalize expression using reference populations

When designing multiparameter panels, researchers should note that STK4 is expressed at varying levels across immune cell subtypes, with high expression in monocytes and T cells but lower levels in neutrophils . Panel design should account for these differences to ensure adequate sensitivity across all populations of interest.