STK24 Antibody

What is STK24 and what cellular processes does it regulate?

STK24, also known as MST3, STK3, MST3B, or STE20, is a serine/threonine kinase that belongs to the GCKIII subfamily. It plays crucial roles in various cellular processes including cell cycle regulation, cell survival, and apoptosis . STK24 is predominantly localized in the cytoplasm and functions as an important signaling molecule in pathways that regulate cell growth and survival .

At the molecular level, STK24 has been identified as a direct regulator of AKT signaling. It associates with and directly phosphorylates AKT at Threonine 21 (Thr21), which promotes AKT activation and subsequent downstream signaling events . This phosphorylation event is particularly significant in cancer biology, as it contributes to tumor progression through various mechanisms including immune evasion.

Recent studies have revealed that STK24 expression is elevated in multiple tumor types compared to adjacent normal tissues, and this elevated expression is inversely correlated with patient survival . This positions STK24 as both a potential prognostic marker and therapeutic target in cancer research.

How does STK24 influence tumor immune evasion mechanisms?

STK24 plays a critical role in tumor immune evasion through several interconnected mechanisms. The most well-characterized pathway involves the regulation of PD-L1 expression on tumor cells. STK24 promotes the expression of PD-L1 through AKT phosphorylation and activation . When PD-L1 engages with PD-1 on immune cells, it suppresses their anti-tumor activity, creating an immunosuppressive tumor microenvironment.

Experimental evidence has demonstrated that deletion or inhibition of STK24 blocks IFN-γ-mediated PD-L1 expression in tumor cells . This has significant implications for anti-tumor immunity, as IFN-γ is widely believed to be the predominant stimulator contributing to inducible PD-L1 expression in the tumor microenvironment.

Flow cytometry analyses have revealed that STK24 deficiency in tumor cells leads to significantly increased infiltration and activation of cytotoxic CD8+ T cells and NK cells in tumor tissues . Importantly, the anti-tumor effect of STK24 deficiency was abrogated when CD8+ T cells or NK cells were depleted, confirming that STK24 supports tumor development through inhibition of CD8+ T cell-dependent and NK cell-dependent cytotoxic responses .

These findings collectively identify STK24 as a critical modulator of antitumor immunity through its ability to regulate PD-L1 expression and shape the immune landscape within tumors.

What are the key characteristics of STK24 antibodies used in research?

STK24 antibodies used in research are designed to detect this important kinase with high specificity and sensitivity. The STK24 Rabbit Polyclonal Antibody (such as CAB10576) is typically generated against specific epitopes of the STK24 protein. For instance, a common immunogen is a recombinant fusion protein containing a sequence corresponding to amino acids 312-431 of human STK24 (NP_001027467.2) .

These antibodies are valuable tools for researchers studying STK24's role in various cellular processes and its implications in cancer. By specifically binding to the STK24 protein, these antibodies allow for accurate detection and analysis in a variety of cell types, making them essential for studies in cell biology and cancer research .

How should Western blot protocols be optimized for STK24 detection?

Optimizing Western blot protocols for STK24 detection requires attention to several key methodological considerations:

• Sample Preparation:

  • Use RIPA buffer supplemented with protease inhibitors and importantly, phosphatase inhibitors (especially when studying STK24's role in phosphorylation events)

  • Ensure complete lysis by incubating on ice for 30 minutes with occasional vortexing

  • Clarify lysates by centrifugation at 12,000g for 15 minutes at 4°C

  • Quantify protein concentration using a reliable method (BCA or Bradford assay)

• Protein Loading and Separation:

  • Load 20-40 μg of total protein per lane

  • Use 10-12% SDS-PAGE gels for optimal separation of STK24 (molecular weight approximately 52 kDa)

  • Include positive control samples such as HeLa, A375, or U-251MG cell lysates

• Transfer and Blocking:

  • Transfer to PVDF or nitrocellulose membranes at 100V for 60-90 minutes in cold transfer buffer

  • Verify transfer efficiency with Ponceau S staining

  • Block membranes in 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Antibody Incubation and Detection:

  • Dilute primary STK24 antibody in blocking buffer at 1:1000 - 1:2000

  • Incubate with primary antibody overnight at 4°C with gentle agitation

  • Wash membranes thoroughly with TBST (4 × 5 minutes)

  • Incubate with appropriate HRP-conjugated secondary antibody at 1:5000 - 1:10000

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Ensure appropriate exposure time to avoid signal saturation

• Controls and Validation:

  • Include appropriate loading controls (β-actin, GAPDH, or tubulin)

  • Consider including STK24 knockdown/knockout samples as negative controls

  • For phosphorylation studies, include phosphatase-treated samples as controls

Following these methodological steps will help ensure specific and reproducible detection of STK24 in Western blot applications, facilitating reliable interpretation of results in the context of cancer and immunology research.

What experimental models are suitable for studying STK24 function in cancer and immunity?

Several experimental models have been validated for studying STK24 function in cancer and immunity, each offering unique advantages for different research questions:

• Cell Line Models:

  • Human cell lines: HeLa, A375, U-251MG, HT-29 (validated for STK24 expression)

  • Mouse cell lines: NIH/3T3, CT26, MC38, LLC, KPC cells (used in functional studies)

  • These models are useful for mechanistic studies, protein-protein interactions, and signaling pathway analyses

• Genetic Manipulation Models:

  • CRISPR-Cas9 knockout systems: Generation of STK24-deficient cell lines

  • siRNA/shRNA knockdown systems: Transient or stable reduction of STK24 expression

  • Overexpression systems: Ectopic expression of wild-type or mutant STK24

  • These approaches allow for precise manipulation of STK24 levels and activity to study functional consequences

• In Vivo Tumor Models:

  • Syngeneic mouse models: Implantation of STK24-manipulated murine cancer cells into immunocompetent mice

  • Models used successfully include:

    • CT26 cells in BALB/c mice (colon cancer model)

    • MC38 cells in C57BL/6 mice (colon cancer model)

    • LLC cells in C57BL/6 mice (lung cancer model)

    • KPC cells in C57BL/6 mice (pancreatic cancer model)

  • These models preserve intact immune systems, essential for studying STK24's role in tumor-immune interactions

• Therapeutic Intervention Models:

  • Antibody blockade studies: Combining STK24 manipulation with anti-PD-1 antibody treatment

  • In vivo siRNA delivery: Lipid-based nanoparticle (LNP) delivery system for STK24 siRNA to tumors

  • Useful for assessing STK24 as a therapeutic target and its interactions with established immunotherapies

• Clinical Sample Analysis:

  • Tissue microarrays (TMAs): For correlative studies of STK24 expression with clinical parameters

  • Multiplexed immunofluorescence: For studying STK24 in relation to immune cell infiltration

  • These approaches bridge preclinical findings with clinical relevance

Experimental evidence gathered from these models has demonstrated that STK24 deficiency in tumor cells suppresses tumor growth by orchestrating infiltration of activated CD8+ T cells and NK cells . These models have been instrumental in establishing STK24 as a critical modulator of antitumor immunity and a potential therapeutic target for cancer immunotherapy.

How does STK24 regulate AKT phosphorylation and what are the implications?

STK24 has been identified as a direct regulator of AKT signaling through a previously unrecognized mechanism. Specifically, STK24 associates with and directly phosphorylates AKT at Threonine 21 (Thr21) . This phosphorylation event promotes AKT activation, which subsequently leads to downstream signaling events, including the induction of PD-L1 expression .

The STK24-AKT-PD-L1 signaling axis has significant implications for cancer biology and immunotherapy:

• Tumor Cell Intrinsic Effects:

  • Enhanced AKT signaling promotes cell survival, proliferation, and metabolic reprogramming

  • These effects contribute to tumor growth and progression independent of immune interactions

• Immune Evasion Mechanisms:

  • Increased PD-L1 expression inhibits T cell and NK cell activation

  • Creates an immunosuppressive tumor microenvironment

  • Facilitates escape from immune surveillance

• Therapeutic Resistance:

  • May contribute to resistance against conventional therapies

  • Provides a mechanistic explanation for variable responses to PD-1/PD-L1 blockade therapies

Quantitative standardized immunohistochemistry analyses have revealed that the expression level of STK24 protein positively correlates with the phosphorylation levels of AKT-T21 and PD-L1 expression in human colorectal, lung, and pancreatic cancer tissues . This relationship has been observed consistently across different tumor types, suggesting a conserved mechanism.

These findings highlight the potential of targeting the STK24-AKT axis as a strategy to enhance anti-tumor immunity and overcome resistance to immunotherapy. Inhibition of STK24 could potentially decrease AKT phosphorylation, reduce PD-L1 expression, and create a more favorable environment for immune-mediated tumor rejection.

How does STK24 deficiency affect immune cell infiltration in tumors?

STK24 deficiency significantly alters the immune landscape within tumors, particularly affecting cytotoxic immune cell populations. Flow cytometry analyses have revealed several key changes in the tumor microenvironment when STK24 is depleted:

• Enhanced CD8+ T Cell Infiltration and Activity:

  • Significantly increased activity (IFN-γ+ or GZMB+) of infiltrated CD8+ T cells in STK24-deficient tumors

  • Increased proportion of active IFN-γ+ CD8+ T cells in STK24-deficient MC38 cell-inoculated mice

  • These activated T cells exhibit enhanced cytotoxic potential against tumor cells

• Increased NK Cell Infiltration and Activity:

  • STK24-deficient LLC tumors showed elevated infiltrations of GZMB+ and IFN-γ+ NK cells compared to wild-type controls

  • Increased proportion of active IFN-γ+ NK cells in tumor tissues with STK24-deficient cells

  • NK cells represent an important component of the anti-tumor response in certain contexts

• Cell Type-Specific Dependency:

  • In CT26 tumor models, the anti-tumor effect of STK24 deficiency was dependent on CD8+ T cells

  • In LLC tumor models, the anti-tumor effect primarily depended on NK cells

  • This suggests context-dependent mechanisms that may vary between tumor types

• Synergy with Immunotherapy:

  • STK24 deficiency combined with anti-PD-1 therapy strongly boosted the intratumoral infiltration and activation of CD8+ T cells and NK cells

  • This synergistic effect enhanced the efficacy of immune checkpoint blockade therapy

Importantly, these findings from preclinical models correlate with observations in human tumors. Immunofluorescence staining has revealed a significant augmentation of GZMB+CD8+ T cells in colorectal cancer and pancreatic cancer patients with low expression of STK24 . Analysis of public databases has further confirmed that STK24 expression negatively correlates with the infiltration of CD8+ T cells or NK cells across multiple tumor types .

These results indicate that STK24 inhibition could potentially transform "cold" tumors with limited immune infiltration into "hot" tumors more amenable to immunotherapy, representing a promising therapeutic strategy.

How can STK24 antibodies be used to analyze patient samples in clinical research?

STK24 antibodies serve as valuable tools for analyzing patient samples in clinical research settings. Several methodological approaches have been validated:

• Immunohistochemistry (IHC) Analysis:

  • STK24 antibodies can be used for IHC staining of formalin-fixed, paraffin-embedded (FFPE) tumor tissues

  • Recommended dilution: 1:50 - 1:200

  • Allows visualization of STK24 protein expression levels and patterns within the tumor microenvironment

  • Can be quantified using standardized scoring systems (e.g., H-score, percentage of positive cells)

  • Has been successfully applied to human colorectal, lung, and pancreatic cancer tissues

• Immunofluorescence (IF) Multiplexing:

  • Enables simultaneous detection of STK24 along with immune cell markers (e.g., CD8, GZMB)

  • Provides spatial information about STK24 expression relative to immune cell infiltration

  • Used to demonstrate inverse correlation between STK24 expression and GZMB+CD8+ T cell infiltration in patient samples

• Tissue Microarray (TMA) Analysis:

  • Allows high-throughput screening of STK24 expression across large patient cohorts

  • Facilitates correlation analyses with clinical parameters and outcomes

  • Has revealed that STK24 expression positively correlates with phosphorylation levels of AKT-T21 and PD-L1 expression in human tumors

• Correlation with Clinical Parameters:

  • STK24 antibody staining can be correlated with:

    • Patient survival data

    • Tumor stage and grade

    • Response to immunotherapy

    • Immune cell infiltration patterns

These methodological approaches enable researchers to translate findings from preclinical models to human cancer biology. For example, studies using STK24 antibodies have shown that elevated STK24 levels in patient specimens across multiple tumor types inversely correlate with intratumoral infiltration of cytotoxic CD8+ T cells and with patient survival . Such findings provide rationale for developing STK24-targeted therapies and for exploring STK24 as a biomarker for patient stratification in immunotherapy trials.

How is STK24 being targeted in immunotherapy research?

STK24 has emerged as a promising target for enhancing cancer immunotherapy, with several innovative approaches under investigation:

• Combination with Immune Checkpoint Blockade:

  • STK24 deficiency synergizes with anti-PD-1 antibody treatment in multiple tumor models

  • This combination has shown efficacy even in tumor types that typically show resistance to immune checkpoint inhibitors (CT26, LLC, and KPC cells)

  • The mechanistic basis involves reduced PD-L1 expression and enhanced immune cell infiltration and activation

• RNA Interference Approaches:

  • Lipid nanoparticle (LNP) delivery of STK24 siRNA has shown efficacy in mouse models

  • In vivo silencing of STK24 inhibits tumorigenesis and significantly enhances anti-PD-1 therapy

  • This approach provides proof-of-principle that STK24 silencing could serve as a therapeutic strategy

• Small Molecule Development:

  • Given that STK24 facilitates tumor immune evasion through its kinase activity, small molecules specifically designed to target STK24 enzyme activity represent a plausible strategy

  • These could be combined with immune checkpoint blockade for enhanced efficacy

  • Still in early development stages in the preclinical setting

• Biomarker Development:

  • STK24 expression or activity could be developed as a biomarker to predict response to immunotherapy

  • STK24 antibodies could be utilized in companion diagnostic assays for patient stratification

  • This could help identify patients most likely to benefit from combination approaches

Experimental evidence has demonstrated that STK24 deletion in tumor cells leads to increased infiltration and activation of CD8+ T cells and NK cells, resulting in attenuated tumor growth . When combined with anti-PD-1 antibody therapy, STK24 deficiency overcomes intrinsic resistance to immunotherapy in several tumor models .

These findings highlight the potential of STK24 as a therapeutic target for enhancing the efficacy of existing immunotherapies and expanding the range of cancers that can be effectively treated with immune-based approaches.

What are the key considerations for quantifying STK24 expression levels?

Accurate quantification of STK24 expression is essential for comparative studies and correlation with biological or clinical parameters. Researchers should consider these methodological approaches:

• Western Blot Quantification:

  • Always normalize STK24 band intensity to a loading control (β-actin, GAPDH, or tubulin)

  • Ensure detection falls within the linear range of the assay by running a standard curve

  • Perform at least three biological replicates for statistical validity

  • Use dedicated image analysis software (ImageJ, Image Lab) with consistent settings

  • Apply appropriate statistical tests for comparing conditions

• Immunohistochemistry Quantification:

  • Use standardized scoring systems:

    • H-score: Combines intensity (0-3) and percentage of positive cells (0-100%)

    • Allred score: Combines intensity (0-3) and proportion score (0-5)

  • Consider digital pathology platforms for objective assessment

  • Account for intratumoral heterogeneity by sampling multiple regions

  • Have multiple observers score slides independently when possible

• Flow Cytometry Quantification:

  • Measure mean fluorescence intensity (MFI) for relative STK24 expression levels

  • Include fluorescence minus one (FMO) controls for accurate gating

  • Use consistent gating strategies across samples

  • Consider multi-parameter analysis to correlate STK24 with other markers

• Transcript-Level Quantification:

  • Use qRT-PCR with normalization to validated reference genes

  • For RNA-Seq analysis, apply appropriate normalization methods (TPM, FPKM)

  • Validate correlation between protein and mRNA levels

  • Consider the potential influence of post-transcriptional regulation

• Considerations for Clinical Correlations:

  • Define clear criteria for "high" vs. "low" expression

  • Consider using quartiles, median split, or optimized cut-points

  • Account for confounding factors in multivariate analysis

  • Validate findings in independent cohorts when possible

By applying these rigorous quantification methods, researchers can generate reliable data on STK24 expression levels that can be meaningfully correlated with experimental conditions or clinical outcomes. This is particularly important when evaluating STK24 as a potential biomarker or therapeutic target in cancer.

How can I reduce background when using STK24 antibodies in immunohistochemistry?

High background is a common challenge in immunohistochemistry that can obscure specific STK24 signals. Here are methodological approaches to reduce background and improve signal-to-noise ratio:

• Blocking Optimization:

  • Use 5-10% normal serum from the same species as the secondary antibody

  • Extend blocking time to 1-2 hours at room temperature

  • Include 0.1-0.3% Triton X-100 or Tween-20 to reduce non-specific binding

  • Test different blocking reagents (BSA, casein, commercial blockers) if standard blocking is insufficient

• Antibody Dilution and Incubation:

  • Perform a dilution series (e.g., 1:50, 1:100, 1:200, 1:400) to identify optimal concentration

  • Use blocking buffer with 0.05-0.1% detergent for antibody dilution

  • Incubate at 4°C overnight rather than at room temperature

  • Increase number and duration of wash steps (e.g., 5 x 5 minutes with gentle agitation)

• Tissue Preparation Considerations:

  • Optimize antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0)

  • For HRP-based detection, block endogenous peroxidase (3% H₂O₂, 10 minutes)

  • For biotin-based detection systems, use avidin/biotin blocking kit

  • For IF, treat sections with 0.1% Sudan Black B or commercial autofluorescence reducers

• Detection System Modifications:

  • Use highly cross-adsorbed secondary antibodies

  • Consider polymeric detection systems for enhanced sensitivity and reduced background

  • Carefully monitor signal development to prevent overdevelopment

  • For fluorescence, select fluorophores with spectral properties that minimize autofluorescence

• Controls and Validation:

  • Include no-primary-antibody controls and isotype controls in each experiment

  • Use known positive tissues (e.g., mouse liver, kidney, lung) as reference standards

  • Consider pre-absorption controls if immunizing peptide is available

By systematically optimizing these parameters, researchers can achieve clean, specific staining of STK24 in tissue sections, enabling accurate assessment of expression patterns and correlations with biological or clinical parameters. This is particularly important when evaluating STK24 as a potential biomarker for cancer progression or response to therapy.