STK36 Antibody

What is STK36 and what biological pathways is it involved in?

STK36 is a serine/threonine protein kinase that plays a crucial role in the sonic hedgehog (Shh) pathway by regulating the activity of GLI transcription factors. It controls the activity of transcriptional regulators GLI1, GLI2, and GLI3 by opposing the effect of SUFU and promoting their nuclear localization. STK36 is also essential for postnatal development, potentially by regulating the homeostasis of cerebral spinal fluid or ciliary function. Recent research has identified its involvement in cancer progression, particularly in prostate cancer where it promotes epithelial-mesenchymal transition (EMT) .

How should I select the appropriate STK36 antibody for my specific research application?

When selecting an STK36 antibody, consider these critical factors:

• Target epitope specificity: Different antibodies target distinct regions of STK36 (N-terminal, C-terminal, or internal regions). For instance, antibodies targeting amino acids 250-450 may be optimal for detecting full-length protein, while those targeting the N-terminus (AA 21-120) might be better for identifying specific isoforms .

• Application compatibility: Verify the antibody has been validated for your specific application:

  • For Western blotting: Most STK36 antibodies are validated (1:1000 dilution is commonly used)

  • For IHC: Select antibodies specifically validated for tissue sections (typically used at 1:100 dilution)

  • For IF/ICC: Ensure the antibody works in immunofluorescence applications

• Species reactivity: Confirm cross-reactivity with your experimental model (human, mouse, rat) .

• Clonality consideration: Polyclonal antibodies offer broader epitope recognition but may have batch variation; monoclonal antibodies provide greater specificity and reproducibility .

What are the expected molecular weight and banding patterns for STK36 in Western blot?

• Protein loading should be optimized at approximately 20μg per lane in SDS-PAGE, as used in validated protocols .

• Expected banding patterns may vary based on:

  • Cell/tissue type (prostate cancer cells like DU-145 and PC-3 show strong expression)

  • Experimental conditions (reducing vs. non-reducing)

  • Post-translational modifications affecting mobility

• For normalization, GAPDH (36 kDa) is commonly used as a loading control at 1:1000 dilution .

Most validated STK36 antibodies show minimal non-specific binding when used at recommended concentrations.

How can I effectively use STK36 antibodies to investigate epithelial-mesenchymal transition in cancer research?

STK36 has been implicated in promoting epithelial-mesenchymal transition (EMT), particularly in prostate cancer. To investigate this relationship:

• Design a comprehensive protein expression analysis panel including:

  • STK36 (1:1000 dilution, primary antibody)

  • E-Cadherin (epithelial marker, 1:1000 dilution)

  • Vimentin (mesenchymal marker, 1:1000 dilution)

  • N-Cadherin (mesenchymal marker, 1:1000 dilution)

  • β-Catenin (EMT regulator, 1:1000 dilution)

  • Slug (EMT transcription factor, 1:1000 dilution)

• Implement parallel experimental approaches:

  • Western blot for protein level quantification

  • Immunofluorescence for subcellular localization

  • IHC for tissue distribution patterns

• Design functional experiments through STK36 manipulation:

  • Overexpression studies to observe EMT marker changes

  • Knockdown/silencing experiments to confirm reversibility of EMT phenotype

  • Treatment with pathway inhibitors to determine mechanism

Research has demonstrated that STK36 upregulation in prostate cancer cells results in decreased E-Cadherin expression and increased Vimentin expression, confirming its role in EMT promotion .

What are the best methodological approaches for studying STK36's role in docetaxel resistance?

To investigate STK36's involvement in docetaxel resistance, implement this methodological framework:

• Cell model development:

  • Establish docetaxel-resistant prostate cancer cell lines (e.g., DU-145 and PC-3) through progressive exposure to increasing docetaxel concentrations

  • Quantify STK36 expression in resistant vs. sensitive cells via Western blot

• Functional assessment through STK36 manipulation:

  • Overexpress STK36 in sensitive cells and test if this confers resistance

  • Silence STK36 in resistant cells to determine if sensitivity is restored

  • Design combination treatments with docetaxel and potential STK36 inhibitors

• Resistance mechanisms evaluation:

  • Measure apoptosis via TUNEL assay following docetaxel treatment in cells with modified STK36 expression

  • Assess proliferation using Cell Counting Kit-8 (CCK8)

  • Evaluate invasion and migration through wound scratch and transwell assays

Research has demonstrated that STK36 overexpression significantly promotes proliferation, invasion, and migration of prostate cancer cells and compensates for the suppression caused by docetaxel treatment in vitro. Additionally, STK36 overexpression inhibits docetaxel-induced apoptosis, suggesting its direct role in treatment resistance .

What controls should I include when using STK36 antibodies in immunohistochemistry of clinical samples?

For rigorous IHC analysis with STK36 antibodies in clinical samples, incorporate these essential controls:

• Technical controls:

  • Positive tissue control: Use prostate cancer tissues known to express high STK36 levels

  • Negative tissue control: Include adjacent normal prostate tissue with low STK36 expression

  • Antibody controls:

    • Primary antibody omission control

    • Isotype control (matched IgG at equivalent concentration)

    • Peptide competition/blocking control

• Analytical approach:

  • Implement digital scanning at high resolution (0.5 mm/pixel using systems like T3 ScanScope)

  • Establish clear scoring criteria (≥5% area positivity is typically considered positive)

  • For tissue microarrays, average scores from multiple cores (typically three) from each patient sample

  • Use streptavidin-horseradish peroxidase complex (1:2000 dilution) for detection

• Validation through correlation:

  • Compare IHC results with mRNA expression data from companion samples

  • Correlate with clinicopathological parameters (Gleason score, tumor grade, pathological pattern)

How can STK36 antibodies be utilized to investigate its role in the Sonic Hedgehog pathway?

To investigate STK36's function in the Sonic Hedgehog (Shh) pathway:

• Protein-protein interaction studies:

  • Co-immunoprecipitation (Co-IP) using STK36 antibodies to identify interactions with:

    • GLI transcription factors (GLI1, GLI2, GLI3)

    • SUFU (Suppressor of Fused)

    • Other pathway components

  • Proximity ligation assay (PLA) to visualize in situ interactions

• Functional analysis of nuclear localization:

  • Immunofluorescence with STK36 antibodies combined with GLI antibodies to assess co-localization

  • Nuclear/cytoplasmic fractionation followed by Western blot to quantify STK36-dependent GLI nuclear translocation

• Signaling pathway activation assessment:

  • Evaluate downstream target gene expression following STK36 manipulation

  • Determine if STK36's effect on GLI factors requires its kinase activity by using kinase-dead mutants

Research shows that STK36 controls the activity of transcriptional regulators GLI1, GLI2, and GLI3 by opposing the effect of SUFU and promoting their nuclear localization. Interestingly, GLI2 requires an additional function of STK36 to become transcriptionally active, but the enzyme does not need to possess an active kinase catalytic site for this to occur .

How do I design experiments to investigate if STK36 is a potential therapeutic target in cancer?

To evaluate STK36 as a therapeutic target, implement this comprehensive experimental design:

What approaches should I use to investigate STK36's role in ciliary development and function?

To study STK36's involvement in ciliary development and function:

• Model systems selection:

  • Primary ciliated cells (e.g., respiratory epithelial cells)

  • Multi-ciliated cells (e.g., ependymal cells lining ventricles)

  • Developmental models (zebrafish, mouse embryos)

• Structural analysis techniques:

  • Immunofluorescence co-localization of STK36 with ciliary markers:

    • Acetylated α-tubulin (axoneme)

    • γ-tubulin (basal bodies)

    • Centrin (centrioles)

  • Super-resolution microscopy to precisely localize STK36 within ciliary structures

  • Electron microscopy to examine central pair apparatus ultrastructure

• Functional assessment:

  • High-speed video microscopy to analyze ciliary beat frequency following STK36 manipulation

  • Flow-based measurements of mucociliary clearance

  • Cerebrospinal fluid dynamics in STK36-deficient models

Research indicates that STK36 is essential for construction of the central pair apparatus of motile cilia and may regulate the homeostasis of cerebral spinal fluid through its effects on ciliary function .

What are the most common technical challenges when using STK36 antibodies and how can they be overcome?

When working with STK36 antibodies, researchers frequently encounter these challenges:

• Background signal issues:

  • For Western blot: Optimize blocking conditions (5% skim milk in PBST for 1 hour at room temperature has proven effective)

  • For IHC/IF: Extend blocking time and consider alternative blocking agents (BSA, serum)

  • Increase washing frequency and duration with PBST

• Detection sensitivity challenges:

  • For low abundance samples: Implement signal amplification methods

  • Optimize protein extraction methods (SDS Lysis Buffer has been validated for STK36)

  • For Western blot, consider using ECL detection systems with appropriate exposure times

• Antibody specificity concerns:

  • Validate with positive and negative control samples

  • Perform peptide competition assays

  • Compare results with multiple antibodies targeting different epitopes of STK36

• Quantification accuracy:

  • For Western blot, normalize to appropriate loading controls (GAPDH at 1:1000 dilution)

  • For IHC, use digital scanning systems with consistent parameters (0.5 mm/pixel resolution)

  • Analyze images using standardized software (e.g., ImageJ for densitometric analysis)

How can I validate the specificity of my STK36 antibody results in experimental systems?

To confirm STK36 antibody specificity, implement this multi-level validation strategy:

• Expression modulation validation:

  • Compare antibody signal in systems with:

    • STK36 overexpression (verify increased signal)

    • STK36 knockdown/knockout (verify decreased/absent signal)

  • Confirm changes via orthogonal methods (e.g., mRNA quantification)

• Cross-platform verification:

  • Compare results across multiple detection methods:

    • Western blot (for protein size verification)

    • IHC/IF (for localization patterns)

    • Flow cytometry (for quantitative assessment)

• Epitope-specific validation:

  • Test multiple antibodies targeting different regions of STK36

  • Conduct epitope mapping or peptide competition assays

  • For critical findings, confirm with antibodies from different manufacturers/clones

• Bioinformatic cross-checking:

  • Verify antibody recognizes conserved regions across species when using in different models

  • Check for potential cross-reactivity with homologous proteins

What methodological considerations are important when studying both protein and functional aspects of STK36?

When investigating both protein expression and functional roles of STK36:

• Protein detection optimization:

  • Select antibodies appropriate for each application (WB, IHC, IF)

  • Consider epitope accessibility in different experimental contexts:

    • For fixed tissues: Epitope retrieval methods may be necessary

    • For native protein studies: Use antibodies recognizing accessible epitopes

• Functional assay design:

  • When studying STK36's role in proliferation: Use CCK8 assays at appropriate time points (24h, 48h, 72h)

  • For migration/invasion studies: Implement both wound healing and transwell assays

  • For apoptosis assessment: TUNEL assay has been validated

• Data integration approaches:

  • Correlate protein expression levels with functional outcomes

  • Implement rescue experiments to confirm specificity of effects

  • Use pathway inhibitors to dissect mechanistic relationships

• Interpretation considerations:

  • Account for cell type-specific functions of STK36

  • Consider potential scaffolding vs. enzymatic roles

  • Distinguish between direct and indirect effects

How should I interpret contradictory results between STK36 protein expression and functional outcomes?

When facing discrepancies between STK36 expression data and functional results:

• Technical evaluation:

  • Verify antibody specificity across different applications

  • Assess whether antibodies detect all relevant isoforms

  • Consider post-translational modifications that might affect function without changing total protein levels

• Biological complexity considerations:

  • STK36 may have kinase-dependent and independent functions

    • Research shows GLI2 requires STK36 function to become transcriptionally active, but the enzyme does not need to possess an active kinase catalytic site

  • Context-dependent roles in different cellular compartments

  • Potential compensation by related kinases

• Methodological approach:

  • Implement domain-specific mutations to dissect functional regions

  • Use phospho-specific antibodies to assess activation status

  • Conduct temporal studies to identify dynamic changes

• Resolution strategies:

  • Single-cell analysis to identify heterogeneous responses

  • Pathway-focused approaches to place contradictions in context

  • Combinatorial inhibition/activation to test redundancy hypotheses

What approaches should I use to correlate STK36 expression with clinical parameters in cancer research?

For rigorous correlation of STK36 expression with clinical parameters:

How can I integrate STK36 research findings with other signaling pathways in comprehensive cancer studies?

To integrate STK36 research within broader cancer signaling networks:

• Pathway intersection analysis:

  • Map STK36 regulation of Hedgehog signaling with:

    • EMT pathways (focus on E-Cadherin, Vimentin, N-Cadherin regulation)

    • Drug resistance mechanisms (particularly docetaxel resistance in prostate cancer)

    • Cancer stemness pathways (often regulated by Hedgehog signaling)

• Multi-omics integration methods:

  • Correlate STK36 protein expression with:

    • Transcriptomic profiles (key EMT gene signatures)

    • Phosphoproteomics (downstream signaling effects)

    • Chromatin accessibility (effects on epigenetic regulation)

• Systems biology approaches:

  • Network analysis to identify key nodes connecting STK36 to other pathways

  • Computational modeling of pathway crosstalk

  • Identification of potential synthetic lethality partners

• Translational application frameworks:

  • Rational combination therapy design based on pathway interactions

  • Biomarker panels including STK36 and related pathway components

  • Patient stratification algorithms incorporating pathway activation status