STK38L Antibody

What is STK38L and what cellular functions does it regulate?

STK38L (serine/threonine kinase 38 like), also known as KIAA0965 and NDR2, belongs to the protein kinase superfamily and AGC Ser/Thr protein kinase family. It is primarily involved in the regulation of structural processes in differentiating and mature neuronal cells . Recent research has demonstrated that STK38L also plays a significant role in immune response regulation, particularly in TLR9-mediated signaling pathways. The protein functions as a negative regulator by constitutively associating with ubiquitin E3 ligase Smurf1, facilitating Smurf1-mediated MEKK2 ubiquitination and degradation .

What are the key specifications of commercially available STK38L antibodies?

STK38L antibodies are available with various specifications to suit different experimental needs. The table below summarizes key characteristics of available antibodies:

The observed molecular weight of STK38L is 54 kDa with 464 amino acids . When selecting an antibody, researchers should consider the specific application requirements and target epitope region.

What experimental applications are supported by STK38L antibodies?

STK38L antibodies can be utilized in multiple experimental applications with varying recommended dilutions:

It is recommended that researchers titrate the antibody in each testing system to obtain optimal results, as performance may be sample-dependent . When performing IHC, suggested antigen retrieval methods include using TE buffer (pH 9.0) or alternatively citrate buffer (pH 6.0) .

How does STK38L regulate TLR9-mediated immune responses in macrophages?

STK38L plays a crucial role in negatively regulating TLR9-mediated immune responses in macrophages through a specific molecular mechanism. Research has shown that STK38L constitutively associates with the ubiquitin E3 ligase Smurf1 and facilitates Smurf1-mediated MEKK2 ubiquitination and degradation . MEKK2 is specifically required for CpG-induced ERK1/2 activation and the production of inflammatory cytokines TNF-α and IL-6, but is not required for LPS-induced production of these cytokines .

The regulatory pathway operates as follows:

• STK38L forms a complex with Smurf1

• This complex targets MEKK2 for ubiquitination

• MEKK2 degradation prevents excessive ERK1/2 activation

• This dampens CpG-induced inflammatory cytokine production

Consequently, STK38L deficiency results in increased CpG-induced ERK1/2 activation and elevated TNF-α and IL-6 production without significantly affecting LPS-induced cytokine production . This selective regulation highlights STK38L's importance in limiting inflammatory responses specifically through TLR9 signaling.

What in vivo effects have been observed in STK38L-deficient models?

STK38L deficiency produces significant in vivo phenotypes related to inflammatory responses. Studies with STK38L-deficient mice have revealed:

• Increased production of pro-inflammatory cytokines TNF-α and IL-6 upon bacterial challenge

• Higher mortality rates following E. coli infection compared to wild-type control mice

• Increased susceptibility to cecal ligation and puncture (CLP)-induced sepsis

These findings suggest that STK38L plays a protective role during infection by limiting excessive inflammatory cytokine production . The protein appears essential for protecting the host from inflammatory injury during infection, possibly through its negative regulation of TLR9 signaling pathways. This makes STK38L a potential therapeutic target for inflammatory conditions characterized by dysregulated cytokine production.

What are the key differences between STK38L (NDR2) and related kinases in the AGC family?

STK38L belongs to the highly conserved NDR/LATS kinase family within the AGC Ser/Thr protein kinase superfamily. While the search results don't provide comprehensive comparative data, it's important to note several distinguishing features:

• STK38L (NDR2) plays a specialized role in neuronal differentiation and structure

• Unlike some related kinases, STK38L exhibits selective regulation of TLR9-mediated immune responses

• STK38L has a molecular weight of 54 kDa with 464 amino acids

• It has specific associations with the ubiquitin-proteasome system through Smurf1 interaction

Understanding these distinctions is crucial for researchers focusing on AGC kinase family members, as experimental approach and interpretation should account for these functional differences.

What are the optimal storage and handling conditions for STK38L antibodies?

Proper storage and handling of STK38L antibodies are essential for maintaining their reactivity and specificity. The recommended conditions include:

What protocol modifications are recommended for optimizing Western blot detection of STK38L?

For optimal Western blot detection of STK38L, researchers should consider the following protocol recommendations:

• Antibody dilution: Use a dilution range of 1:500-1:2000 for primary antibody (12697-1-AP)

• Positive control selection: HEK-293 cells have been validated as a positive control for STK38L detection

• Sample preparation: Ensure complete cell lysis and protein denaturation for proper exposure of the STK38L epitope

• Detection system: Select an appropriate secondary antibody system compatible with the host species (rabbit for 12697-1-AP and ABIN360006; mouse for BMR00770)

• Blocking optimization: Use a blocking solution that minimizes background while preserving specific signal

For detailed step-by-step protocols, both Proteintech and Iwai North America provide downloadable protocol documents specific to their STK38L antibodies . Researchers should consider titrating the antibody concentration in their specific experimental system to achieve optimal signal-to-noise ratio.

What controls and validation steps should be included when studying STK38L function in immune regulation?

When investigating STK38L's role in immune regulation, several critical controls and validation steps should be incorporated:

• Antibody specificity validation:

  • Western blot verification of antibody specificity using positive controls (e.g., HEK-293 cells)

  • Comparison with STK38L-knockout or knockdown samples

  • Peptide competition assays to confirm epitope specificity

• Functional validation experiments:

  • Compare CpG-induced versus LPS-induced cytokine production to confirm TLR9-specific effects

  • Measure both ERK1/2 phosphorylation and downstream cytokine production (TNF-α and IL-6)

  • Include Smurf1 and MEKK2 expression analyses to confirm the proposed regulatory mechanism

• In vivo model considerations:

  • Include appropriate wild-type controls matched for genetic background, age, and sex

  • Monitor multiple parameters during infection models, including cytokine levels, bacterial load, and survival

How can researchers address inconsistent STK38L detection in Western blot experiments?

Inconsistent STK38L detection in Western blots may stem from several factors. Here are methodological approaches to troubleshoot common issues:

• Sample preparation issues:

  • Ensure complete protein extraction using appropriate lysis buffers

  • Validate protein concentration using reliable quantification methods

  • Use fresh protease inhibitors to prevent degradation

  • Confirm proper sample denaturation (avoid excessive heating which may cause aggregation)

• Antibody-specific considerations:

  • Verify antibody expiration dates and storage conditions

  • Test different antibody dilutions (1:500, 1:1000, 1:2000)

  • Consider using alternative STK38L antibodies targeting different epitopes

  • For 12697-1-AP, positive signal has been confirmed in HEK-293 cells

• Detection system optimization:

  • Increase primary antibody incubation time (overnight at 4°C)

  • Validate secondary antibody compatibility and dilution

  • Consider enhanced chemiluminescence (ECL) reagents with appropriate sensitivity

Researchers should methodically adjust each parameter while maintaining appropriate controls to identify the source of inconsistency in STK38L detection.

What potential cross-reactivity issues should researchers be aware of when using STK38L antibodies?

When using STK38L antibodies, researchers should be vigilant about potential cross-reactivity, particularly with related kinases in the NDR/LATS family. Consider these approaches to address cross-reactivity concerns:

• Potential sources of cross-reactivity:

  • Other AGC family kinases with structural similarity

  • NDR1/STK38, which shares significant homology with STK38L/NDR2

  • Splice variants of STK38L

• Verification approaches:

  • Validate specificity using STK38L-knockout or siRNA-treated samples

  • Perform peptide competition assays with the immunizing peptide

  • Consider testing multiple antibodies targeting different epitopes

  • The 12697-1-AP antibody is reported to be specific to STK38L

• Experimental design considerations:

  • Include negative control tissues/cells known not to express STK38L

  • When possible, corroborate protein detection with gene expression data

  • For critical experiments, consider orthogonal detection methods (e.g., mass spectrometry)

Understanding the antibody's target epitope region (e.g., C-terminal for ABIN360006 ) can help predict potential cross-reactivity based on sequence homology with other proteins.

How do differences in STK38L expression across cell types and tissues impact experimental design?

STK38L expression varies across different cell types and tissues, which has important implications for experimental design:

• Tissue-specific considerations:

  • STK38L is involved in neuronal differentiation and structure, suggesting significant expression in neural tissues

  • Positive IHC detection has been reported in human heart tissue using 12697-1-AP antibody

  • Macrophages express functionally relevant levels of STK38L for immune regulation studies

• Experimental design adaptations:

  • Select appropriate positive controls based on known expression patterns

  • Adjust antibody concentrations for tissues with different expression levels

  • For IHC, antigen retrieval methods may need optimization per tissue type (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Consider the impact of cell activation states on STK38L expression

• Quantification approaches:

  • Use quantitative techniques (qPCR, quantitative Western blot) to determine relative expression levels

  • Normalize protein detection to appropriate housekeeping proteins for the specific tissue type

  • Consider establishing a standard curve using recombinant STK38L for absolute quantification

By accounting for tissue-specific expression patterns and adjusting protocols accordingly, researchers can generate more reliable and reproducible data when studying STK38L across different biological contexts.

What are the emerging roles of STK38L in pathological conditions beyond immune regulation?

While current research has established STK38L's role in immune regulation, its functions likely extend to other pathological conditions that warrant investigation:

• Neurological disorders:

  • Given STK38L's involvement in "regulation of structural processes in differentiating and mature neuronal cells" , it may play roles in neurodevelopmental or neurodegenerative disorders

  • Research could examine STK38L expression and activity in models of neurological diseases

• Cancer biology:

  • As a kinase involved in cellular signaling, STK38L may influence cancer cell proliferation, survival, or migration

  • Studies could investigate whether STK38L expression correlates with specific cancer types or outcomes

• Infectious disease susceptibility:

  • The finding that "Stk38-deficient mice are more susceptible to CLP-induced sepsis" suggests broader roles in infection response

  • Further research could explore STK38L polymorphisms in relation to infectious disease susceptibility

Methodologically, these investigations would benefit from comprehensive approaches combining genetic models, pharmacological manipulations, and clinical sample analyses to establish causative relationships between STK38L function and disease pathology.

How might therapeutic targeting of the STK38L pathway impact inflammatory disorders?

The negative regulatory role of STK38L in TLR9-mediated immune responses suggests potential therapeutic applications in inflammatory disorders:

• Potential therapeutic strategies:

  • STK38L activators could dampen excessive TLR9-mediated inflammation

  • Targeting the STK38L-Smurf1-MEKK2 axis might provide selective modulation of specific inflammatory pathways

  • Combination approaches targeting multiple points in this pathway could enhance therapeutic efficacy

• Disease contexts for investigation:

  • Autoimmune conditions with TLR9 involvement (e.g., systemic lupus erythematosus)

  • Sepsis, where STK38L-deficient mice show increased susceptibility

  • Inflammatory conditions with dysregulated cytokine production

• Methodological considerations for therapeutic development:

  • Development of high-throughput screening assays for STK38L modulators

  • Design of cell-based systems to evaluate pathway-specific effects

  • In vivo models that recapitulate relevant disease features for preclinical validation

The selective nature of STK38L's effects on TLR9 but not TLR4 signaling suggests potential for developing targeted anti-inflammatory therapies with fewer side effects than broad immunosuppressants.

What technical advances are needed to better study STK38L kinase activity in complex biological systems?

Advancing our understanding of STK38L function will require technical innovations to address current methodological limitations:

• Development of specific activity assays:

  • Phospho-specific antibodies to directly monitor STK38L activation state

  • FRET-based biosensors to visualize STK38L activity in live cells

  • Development of selective STK38L inhibitors as research tools

• Systems biology approaches:

  • Proteomics to comprehensively identify STK38L substrates

  • Phosphoproteomics to map STK38L-dependent signaling networks

  • Integration of transcriptomic, proteomic, and functional data to build predictive models

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize STK38L localization at subcellular structures

  • Live cell imaging to track STK38L dynamics during cellular processes

  • Correlative light and electron microscopy to link STK38L function to ultrastructural features