STK26 Antibody

What is STK26 and what cellular functions does it regulate?

STK26 (Sterile 20-like kinase 26), also known as MST4, is a serine/threonine protein kinase belonging to the Ste20 family. It primarily localizes to the Golgi apparatus through binding with the GM130 protein and undergoes autophosphorylation at the Thr178 site. STK26 functions as a regulatory kinase involved in multiple cellular processes including cell proliferation, differentiation, apoptosis, cytoskeleton reorganization, and autophagy .

The protein contains an amino-terminal kinase domain and a carboxy-terminal regulatory domain that mediates homodimerization. Research has demonstrated that STK26 regulates various cellular mechanisms, including type I interferon production through mitochondrial antiviral signaling proteins and direct phosphorylation of β-catenin at Thr40. Additionally, STK26 plays a role in immune cell polarization in certain pathological conditions such as primary immune thrombocytopenia .

How does STK26 expression vary across cancer types and normal tissues?

STK26 expression patterns show considerable variation across cancer types, with significant upregulation observed in multiple malignancies. Analysis using the TCGA database reveals elevated STK26 expression in breast cancer, colorectal adenocarcinoma, cholangiocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, hepatocellular carcinoma (HCC), lung squamous cell carcinoma, and gastric adenocarcinoma compared to corresponding normal tissues .

In hepatocellular carcinoma specifically, STK26 shows consistent overexpression in both unpaired and paired tissue analyses. The promoter methylation levels of STK26 are significantly elevated in HCC compared to normal tissues, suggesting epigenetic regulation as a potential mechanism for its aberrant expression. Western blot analyses of various HCC cell lines confirm elevated STK26 protein levels in most HCC lines compared to normal human liver cells (THLE-2), with the exception of the Li-7 cell line .

What is the calculated versus observed molecular weight of STK26?

The difference between theoretical and observed molecular weights may result from post-translational modifications such as phosphorylation, which STK26 undergoes as part of its activation process. When performing Western blot analysis, researchers should be aware of this potential size difference to accurately identify STK26 bands. The active form of STK26 requires autophosphorylation at Thr178, which can influence protein migration patterns in gel electrophoresis .

What are the validated applications for STK26 antibodies?

STK26 antibodies have been validated for multiple experimental applications, with the primary validated techniques being Western blot (WB), Flow Cytometry (FCM), and Enzyme-Linked Immunosorbent Assay (ELISA) .

For Western blot applications, STK26 antibodies have been successfully used at concentrations of 0.25-0.5 μg/ml for detecting the protein in human and rat samples. Validated positive controls include human HeLa, 293T, and Jurkat whole cell lysates, as well as rat PC-12 whole cell lysates. In flow cytometry applications, antibody concentrations of 1-3 μg per 10^6 cells have shown effective detection in human cell lines such as MCF-7. For ELISA applications, recommended concentrations range from 0.1-0.5 μg/ml .

How does STK26 regulate autophagy in cancer cells?

STK26 plays a crucial role in autophagy regulation, particularly through its interaction with and phosphorylation of ATG4B, a cysteine protease essential for autophagosome formation. The mechanism involves STK26-mediated phosphorylation of ATG4B, which enhances ATG4B's enzymatic activity in processing LC3, a key component of autophagosome membranes .

In hepatocellular carcinoma cells, STK26 enhances ATG4B phosphorylation, increases LC3BII expression, and reduces P62 levels, collectively promoting autophagy. Experimental evidence demonstrates that STK26 knockdown significantly reduces autophagic flux in HCC cells, while its overexpression enhances it. This regulatory mechanism appears to contribute to cancer cell survival and potentially to therapeutic resistance. In glioblastoma, for instance, STK26 promotes autophagy that can reduce radiosensitivity, suggesting that STK26 inhibition might enhance the effectiveness of radiation therapy .

What is the role of STK26 in modulating drug sensitivity in cancer?

STK26 significantly impacts cancer drug sensitivity, particularly through its regulation of autophagy. In hepatocellular carcinoma, STK26-mediated autophagy contributes to sorafenib resistance, a critical clinical challenge in HCC treatment. Research indicates that autophagy induction often serves as a protective mechanism enabling cancer cells to survive drug treatment stress .

Experimental data demonstrates that inhibition of STK26 can enhance sorafenib-induced HCC cell death by suppressing protective autophagy. The mechanism involves the STK26-ATG4B axis, where STK26 activates ATG4B through phosphorylation, enhancing autophagic flux. Additionally, microRNA-22-3p (miR-22-3p) can target both STK26 and ATG4B, thereby inhibiting autophagy and significantly enhancing sorafenib-induced HCC cell death. This suggests a potential therapeutic strategy combining STK26 inhibition with existing treatments to overcome drug resistance in HCC and potentially other cancers .

What methods should be used to validate STK26 antibody specificity?

Validating STK26 antibody specificity requires a multi-faceted approach to ensure experimental results accurately reflect STK26 biology. The following methodological steps are recommended:

• Knockout/knockdown controls: Generate STK26 knockdown cell lines using shRNA or siRNA approaches. Western blot analysis should show reduced or absent STK26 signal in these cells compared to controls. The search results indicate successful creation of stable cell lines with STK26 knockdown using lentiviral particles containing pLKO.1-sh-STK26-puro .

• Overexpression controls: Perform parallel experiments with STK26 overexpression (e.g., using pcDNA3.1-STK26 plasmids as described in the search results) to verify increased antibody signal intensity .

• Cross-reactivity testing: Test the antibody against related kinases, particularly other members of the Ste20 family, to ensure specificity. High-quality antibodies like the one in the search results should show "No cross-reactivity with other proteins" .

• Multiple detection methods: Validate specificity across multiple techniques (Western blot, immunofluorescence, flow cytometry) to ensure consistent results across platforms .

• Multiple cell lines: Test antibody performance across different cell types known to express STK26 at varying levels. The search results validate STK26 detection in various human cell lines (HeLa, 293T, Jurkat) and rat cells (PC-12) .

What are the optimal conditions for detecting STK26 via Western blot?

Based on validated protocols, the following conditions are optimal for STK26 detection by Western blot:

These conditions have been validated for human samples (HeLa, 293T, Jurkat whole cell lysates) and rat samples (PC-12 whole cell lysates) .

How should researchers approach studying STK26's role in cancer progression?

Investigating STK26's role in cancer progression requires a comprehensive experimental approach:

What methods should be used to study STK26-mediated autophagy?

Studying STK26-mediated autophagy requires specialized techniques:

• Autophagic flux assessment: Utilize the mRFP1-EGFP-LC3B reporter system to visualize and quantify autophagosome and autolysosome formation. The search results describe establishing stable cell lines expressing this dual-fluorescent reporter, allowing measurement of autophagic flux through fluorescence microscopy .

• Autophagy marker analysis: Monitor key autophagy markers by Western blot, including:

  • LC3B-I to LC3B-II conversion (increased during autophagy)

  • P62/SQSTM1 levels (decreased during autophagy)

  • ATG4B phosphorylation status (enhanced by STK26)

• STK26-ATG4B interaction studies:

  • Co-immunoprecipitation to confirm physical interaction

  • In vitro kinase assays to assess STK26's ability to phosphorylate ATG4B

  • Site-directed mutagenesis of potential phosphorylation sites to identify key regulatory residues

• Pharmacological modulation: Compare STK26 knockdown/overexpression effects with known autophagy modulators:

  • Rapamycin (inducer)

  • Chloroquine/Bafilomycin A1 (inhibitors of autolysosome formation)

• Functional consequences: Assess how STK26-mediated autophagy affects:

  • Cell survival under stress conditions

  • Drug resistance (particularly to sorafenib in HCC)

  • Cancer cell metabolism and energy homeostasis

How can researchers effectively use STK26 antibodies in flow cytometry?

Flow cytometry with STK26 antibodies requires specific methodological considerations:

• Sample preparation: For fixed cell analysis, use 1-3 μg of antibody per 10^6 cells. The search results validate this approach in human MCF-7 cells .

• Fixation and permeabilization: Since STK26 is primarily an intracellular protein localized to the Golgi apparatus, proper cell permeabilization is crucial. Recommended protocols include:

  • Fixation with 4% paraformaldehyde for 10-15 minutes

  • Permeabilization with 0.1-0.5% Triton X-100 or saponin-based buffers

• Controls:

  • Include isotype controls (rabbit IgG at the same concentration)

  • Include positive controls (e.g., MCF-7 cells as validated in the search results)

  • Include negative controls (cells with STK26 knockdown)

• Gating strategy:

  • Exclude cell debris and doublets

  • Use forward and side scatter to identify viable cells

  • Apply compensation if using multiple fluorochromes

• Analysis considerations:

  • Measure both percentage of positive cells and mean fluorescence intensity

  • Consider subcellular localization patterns in imaging flow cytometry

  • Correlate with Western blot results for validation

What are common issues when detecting STK26 in Western blot and how to resolve them?

Several challenges may arise when detecting STK26 via Western blot:

• Multiple bands/non-specific binding:

  • Issue: Detection of bands other than the expected 52 kDa STK26 band.

  • Solution: Optimize antibody concentration (use 0.25-0.5 μg/ml as validated in the search results), increase blocking time/concentration, and use more stringent washing conditions with TBS-0.1% Tween .

• Weak or no signal:

  • Issue: Inability to detect STK26 despite known expression.

  • Solution: Ensure sufficient protein loading (30 μg of whole cell lysate as recommended), optimize transfer conditions (150 mA for 50-90 minutes), and consider longer exposure times during detection .

• Discrepancy in molecular weight:

  • Issue: STK26 band appears at a different size than the expected 52 kDa.

  • Solution: Be aware that post-translational modifications may alter migration patterns. The calculated molecular weight of 46.529 kDa differs from the observed 52 kDa in validated Western blots .

• Inconsistent results across cell lines:

  • Issue: Variable detection of STK26 in different cell types.

  • Solution: Adjust protein loading based on known expression levels. The search results indicate variable expression across HCC cell lines, with some showing higher expression than others .

• High background:

  • Issue: Non-specific staining obscuring specific STK26 signal.

  • Solution: Use high-quality antibodies designated as "Picoband" that guarantee "superior quality, high affinity, and strong signals with minimal background in Western blot applications" .

How can researchers effectively study the relationship between STK26 and miRNAs?

The relationship between STK26 and microRNAs, particularly miR-22-3p, represents an important regulatory mechanism:

• Luciferase reporter assays:

  • Clone the 3′-UTR sequences of STK26 containing predicted miRNA binding sites into reporter vectors (e.g., pmirGLO as described in the search results)

  • Include both wild-type and mutated binding site versions to confirm specificity

  • Co-transfect with miRNA mimics or inhibitors and measure luciferase activity after 48 hours

• Expression correlation studies:

  • Analyze the inverse correlation between miRNA expression and STK26 protein/mRNA levels

  • Perform qRT-PCR for miRNA quantification alongside Western blot for STK26 protein detection

• Functional rescue experiments:

  • Design experiments that co-express both miRNA (e.g., miR-22-3p) and STK26 to determine if STK26 overexpression can reverse miRNA-induced phenotypes

  • The search results describe such rescue experiments showing that miR-22-3p overexpression significantly inhibited HCC cell proliferation, migration, and invasion by suppressing STK26 expression

• Autophagy regulation assessment:

  • Evaluate how miRNA-mediated STK26 regulation affects autophagy markers

  • The search results show that miR-22-3p overexpression inhibited STK26-induced changes in autophagy-related protein expression and alterations in autophagosome and autolysosome numbers

What emerging technologies might advance STK26 research?

Several cutting-edge technologies hold promise for advancing STK26 research:

• CRISPR-Cas9 genome editing:

  • Generate complete STK26 knockout cell lines and animal models

  • Create knock-in models with tagged STK26 for live-cell imaging

  • Introduce specific mutations to study phosphorylation sites important for STK26 function

• Proximity labeling approaches:

  • BioID or APEX2 fusions with STK26 to identify proximal interacting proteins in living cells

  • Particularly valuable for mapping STK26's interactome at the Golgi apparatus

• Single-cell analysis:

  • Single-cell RNA-seq to identify cell populations with differential STK26 expression

  • Single-cell proteomics to correlate STK26 protein levels with cellular phenotypes

• Advanced imaging techniques:

  • Super-resolution microscopy to precisely localize STK26 within subcellular compartments

  • Live-cell imaging of fluorescently tagged STK26 to track its dynamics during cellular processes

• Therapeutic targeting approaches:

  • Development of small molecule inhibitors specific to STK26

  • Evaluation of miRNA-based therapeutics targeting STK26, particularly miR-22-3p as identified in the search results