stk10 Antibody

What is STK10 and why is it relevant to research?

STK10, also known as lymphocyte-oriented kinase (LOK) or PRO2729, is a serine/threonine kinase belonging to the STE Ser/Thr protein kinase family. The canonical human protein consists of 968 amino acid residues with a calculated molecular weight of 112 kDa, though it typically appears at approximately 130 kDa in western blots . STK10 is predominantly expressed in lymphoid organs including the spleen, thymus, and bone marrow, with significant expression also observed in rapidly proliferating tissues such as placenta and peripheral blood leukocytes . Its relevance to research stems from its involvement in lymphocyte migration, tumor growth regulation, and its emerging role in cancer pathogenesis across multiple tumor types .

The storage and handling recommendations for STK10 antibodies vary by formulation:

• Standard preparations: Store at -20°C with stability for approximately one year after shipment. Aliquoting is generally unnecessary for -20°C storage. Small volume preparations (20μl) may contain 0.1% BSA .

• PBS-only formulations: More stringent storage at -80°C is recommended to maintain antibody integrity .

• Glycerol-containing formulations: These typically contain PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) and offer improved stability during freeze-thaw cycles .

When working with any STK10 antibody preparation, avoid repeated freeze-thaw cycles, keep on ice during experiments, and centrifuge briefly before use to ensure homogeneity.

How can researchers validate STK10 knockout models?

Validation of STK10 knockout models requires a multi-faceted approach:

• Genomic verification: Confirm knockout-induced mutations using DNA sequencing with specific primers. For example, sequencing primers like 5'-GTGCTCCGAAACAGGGC-3' have been successfully used to identify genomic changes in STK10 knockout cell lines .

• Protein expression assessment: Western blot analysis with anti-STK10 antibodies remains the gold standard. Researchers have successfully used multiple antibodies including anti-STK10 (abcam, ab70484) and Proteintech's 25471-1-AP to confirm complete abolishment of STK10 expression in knockout lines .

• Functional validation: Assess known STK10-dependent phenotypes, such as changes in ERM protein phosphorylation, cell migration capacity, or p38 MAPK pathway activation .

• Single-clone isolation: For cellular models, limiting dilution methods (10 cells/ml diluted into 96-well plates at 100 μl/well) have been effectively used to select single clones after puromycin selection .

What are the optimal conditions for STK10 detection in IHC applications?

For optimal STK10 detection in immunohistochemistry applications:

• Antigen retrieval: Use TE buffer pH 9.0 as the primary method. Alternatively, citrate buffer pH 6.0 has also proven effective in some tissue types .

• Antibody concentration: A dilution range of 1:50-1:500 is recommended, with specific optimization required for each tissue type .

• Positive control tissues: Human lymphoma tissue has been validated as an appropriate positive control for STK10 IHC applications .

• Detection systems: Both DAB-based chromogenic and fluorescence-based detection systems have been successfully employed, with the choice depending on the experimental requirements for sensitivity and multiplexing.

• Counterstaining: Standard hematoxylin counterstaining provides adequate nuclear contrast when using chromogenic detection methods.

How does STK10 function differ across cancer types?

Research has revealed context-dependent roles of STK10 across different cancer types:

These divergent effects highlight the tissue-specific nature of STK10 function and suggest that interpretation of STK10 expression must consider the specific cellular context .

What role does STK10 play in the tumor microenvironment?

STK10 exhibits significant associations with the tumor microenvironment (TME), particularly in immune regulation:

• Immune cell correlation: Bioinformatic analyses have shown that STK10 expression is significantly positively associated with tumor-infiltrating immune cells, including lymphocytes, neutrophils, macrophages, and dendritic cells .

• CTL regulation: In STK10 knockout mouse models, tumors show decreased activated/effector cytotoxic T lymphocytes (CTLs) in the TME, suggesting STK10's role in maintaining anti-tumor T-cell responses .

• Angiogenesis modulation: Host STK10 deficiency results in increased vessel density within tumors, indicating a regulatory role in tumor angiogenesis .

• Expression pattern: According to Human Protein Atlas data, STK10 is broadly expressed in immune cells such as NK cells, dendritic cells, and T cells, further supporting its immunoregulatory functions .

These findings suggest that STK10 functions as a critical modulator of anti-tumor immunity by affecting both lymphocyte activation and the vascular microenvironment.

How does STK10 affect ERM proteins and cell migration?

STK10 serves as an ERM (Ezrin, Radixin, Moesin) kinase with complex effects on cell migration:

• Phosphorylation relationship: STK10 directly phosphorylates ERM proteins at critical regulatory sites (ezrin Thr567, radixin Thr564, and moesin Thr558) .

• Context-dependent effects: In prostate cancer cells, STK10 knockout suppresses cell migration primarily through inhibiting ERM protein activation . Conversely, in cervical cancer cells, STK10 deletion promotes migration through ERM-independent mechanisms .

• Detection methods: Anti-ezrin (pThr567)/radixin (pThr564)/moesin (pThr558) antibodies can be used to monitor the direct phosphorylation activity of STK10 on ERM proteins, providing a functional readout of STK10 activity .

This complex relationship suggests that STK10's effects on cell motility are likely determined by the underlying cellular context and additional signaling networks that vary across tissue types.

What are the most effective strategies for generating STK10 knockout models?

CRISPR-Cas9 gene editing has emerged as the preferred method for STK10 knockout generation:

• Guide RNA design: Several effective guide RNA sequences have been validated:

  • 5'-CACCGGCGGACGTGCTCATATTCG-3' and 5'-AAACCGAATATGAGCACGTCCGCC-3' (annealed oligonucleotides for prostate cancer cells)

  • 5'-CACCGACTCACCTCCAGCAGCTGCG-3' and 5'-CACCGCATGATTGAGTTCTGTCCAG-3' (for MV411 cells)

• Vector systems: The PX459 plasmid (Addgene, #62988) has been successfully employed for STK10 knockout in multiple studies .

• Selection protocol: A two-phase puromycin selection protocol (typically 1-2 μg/mL) has proven effective:

  • Initial selection for 1-7 days following transfection

  • Single-cell cloning by limiting dilution

  • Clone screening by Western blot and DNA sequencing

• Verification primers: Sequencing primers such as 5'-GTGCTCCGAAACAGGGC-3' or 5'-CGGGTCTGGGGAGAACCCCG-3' have been utilized for confirmation of genomic changes .

For researchers preferring non-CRISPR approaches, lentiviral shRNA systems have also demonstrated efficacy, though with potential off-target considerations that require more extensive validation controls.

How can researchers differentiate between STK10 expression and activity?

Distinguishing between STK10 expression and its functional activity requires complementary approaches:

• Expression assessment:

  • Western blotting with STK10-specific antibodies (observed at ~130 kDa)

  • qRT-PCR analysis using validated primers for STK10 mRNA

  • IHC staining in tissue sections with optimized protocols

• Activity measurement:

  • Phosphorylation of known substrates (ERM proteins) using phospho-specific antibodies

  • Downstream pathway activation analysis (p38 MAPK pathway components)

  • Functional readouts including cell migration, adhesion, and proliferation assays

• Direct kinase assays:

  • In vitro kinase assays using immunoprecipitated STK10 and recombinant substrates

  • ATP consumption assays to quantify kinase activity

This multi-parameter approach provides a more comprehensive understanding of both STK10 abundance and functional status in experimental systems.

What experimental artifacts or challenges may arise when studying STK10?

Researchers should be aware of several potential challenges when investigating STK10:

• Molecular weight discrepancy: While the calculated molecular weight of STK10 is 112 kDa, it consistently appears at approximately 130 kDa in Western blots, likely due to post-translational modifications. Researchers should account for this difference when interpreting results .

• Cell type-specific effects: As demonstrated across multiple studies, STK10 functions can vary dramatically between cell types, necessitating careful selection of appropriate experimental models and cautious extrapolation of findings .

• Compensation mechanisms: In knockout models, compensatory upregulation of related kinases may occur, potentially masking phenotypes. Comprehensive analysis of related kinase family members is advisable .

• Antibody specificity: Confirming antibody specificity using knockout controls is essential, particularly given STK10's membership in the larger STE20/PAK kinase family with potential cross-reactivity .

• Temporal dynamics: STK10's role in processes like cell migration and immune responses may vary temporally, requiring time-course analyses rather than single time-point measurements .

How might STK10 function as a therapeutic target?

The therapeutic potential of STK10 appears highly context-dependent:

• Cancer-specific considerations: Given STK10's opposing roles in different cancer types, therapeutic targeting strategies would need to be cancer-specific:

  • In prostate cancer, STK10 activation might inhibit proliferation but potentially enhance migration

  • In cervical cancer, STK10 inhibition might reduce migration and invasion capabilities

• Immune modulation potential: STK10's role in regulating CTL activity and tumor angiogenesis suggests potential applications in cancer immunotherapy, possibly through enhancing anti-tumor immune responses .

• Combination approaches: Research indicates potential synergistic effects when combining STK10 inhibition with immunogenic cell death (ICD) inducers, suggesting value in multi-target therapeutic strategies .

While clear therapeutic windows require further definition, the dual roles of STK10 in both tumor cells and immune compartments make it an intriguing target for precision medicine approaches that consider both tumor type and immune context.

What are the emerging methodologies for studying STK10 in complex biological systems?

Advanced techniques that show promise for STK10 research include:

• Single-cell analysis: Given STK10's differential expression across immune populations, single-cell RNA sequencing and protein analysis can provide more nuanced insights into cell-specific functions and expression patterns .

• Spatial transcriptomics: For understanding STK10's role in the tumor microenvironment, spatial methodologies that preserve tissue architecture while enabling molecular profiling will be valuable .

• Phosphoproteomics: Mass spectrometry-based approaches to identify the complete set of STK10 substrates beyond the known ERM proteins will expand our understanding of its signaling networks .

• Inducible knockout systems: Rather than constitutive knockouts, inducible systems allow for temporal control of STK10 deletion, enabling studies of acute versus chronic adaptation to STK10 loss .

• Humanized mouse models: For immunological studies, humanized mouse models may better recapitulate the complexity of human immune responses in STK10-mediated processes .

These emerging methodologies promise to address current knowledge gaps regarding STK10's complex roles in different physiological and pathological contexts.