Phospho-MAP3K5 (Ser83) Antibody

What is MAP3K5/ASK1 and what cellular pathways does it regulate?

MAP3K5 (Mitogen-activated protein kinase kinase kinase 5), also known as ASK1 (Apoptosis signal-regulating kinase 1), is a serine/threonine kinase that functions as an essential component of the MAP kinase signal transduction pathway. It mediates crucial signaling for cellular responses to environmental changes and determination of cell fate, including differentiation and survival. MAP3K5/ASK1 plays a critical role in the apoptosis signal transduction pathway through mitochondria-dependent caspase activation . It serves as a key regulator in the innate immune response, which is essential for host defense against a wide range of pathogens. Additionally, MAP3K5 mediates signal transduction of various stressors including oxidative stress and receptor-mediated inflammatory signals such as tumor necrosis factor (TNF) and lipopolysaccharide (LPS) .

When activated, MAP3K5/ASK1 functions as an upstream activator of two major signal transduction cascades:

• The MKK/JNK (MAP kinase kinase/c-Jun N-terminal kinase) cascade

• The p38 MAPK signal transduction cascade

This activation occurs through phosphorylation of several MAP kinase kinases including MAP2K4/SEK1, MAP2K3/MKK3, MAP2K6/MKK6, and MAP2K7/MKK7, which in turn activate JNKs and p38 MAPKs to control transcription factors like activator protein-1 (AP-1) .

What is the significance of Serine 83 phosphorylation in MAP3K5/ASK1 regulation?

Phosphorylation at Serine 83 (Ser83) represents a critical post-translational modification that regulates MAP3K5/ASK1 activity and function. This specific modification site is located within the N-terminal region (amino acids 1-100) of the protein . Ser83 phosphorylation serves as a regulatory mechanism that modulates the kinase activity of MAP3K5/ASK1 in response to various cellular stimuli and stress conditions.

The state of Ser83 phosphorylation can determine whether MAP3K5/ASK1 activates downstream signaling cascades that lead to cell survival or apoptosis. This phosphorylation event represents one of several regulatory mechanisms that ensure appropriate cellular responses to environmental stressors and signaling inputs. Understanding the dynamics and regulation of Ser83 phosphorylation provides critical insights into how cells coordinate stress responses and maintain homeostasis under challenging conditions.

How does Ser83 phosphorylation differ from other phosphorylation sites on MAP3K5/ASK1?

MAP3K5/ASK1 contains multiple phosphorylation sites that collectively form a complex regulatory network controlling its activity and function. While Ser83 is a key regulatory site, other phosphorylation sites such as Ser966 serve distinct regulatory functions . The specific combination of phosphorylated residues creates a phosphorylation signature that determines MAP3K5/ASK1 activity status and downstream signaling outcomes.

Different phosphorylation sites may respond to different upstream signals and kinases, enabling MAP3K5/ASK1 to integrate multiple inputs from various signaling pathways. The temporal dynamics of phosphorylation at these sites can also differ, allowing for precise temporal control of MAP3K5/ASK1 activity during cellular responses to stress and other stimuli.

What are the primary applications for Phospho-MAP3K5 (Ser83) antibodies?

Phospho-MAP3K5 (Ser83) antibodies serve as essential tools for investigating the phosphorylation state of MAP3K5/ASK1 in various experimental contexts. Based on manufacturer specifications and research literature, these antibodies are validated for multiple applications:

• Western Blot (WB): Enables detection and semi-quantitative analysis of Ser83-phosphorylated MAP3K5 in cell or tissue lysates, with recommended dilutions typically ranging from 1:500 to 1:2000 .

• Immunohistochemistry (IHC): Allows visualization of the spatial distribution of phosphorylated MAP3K5 in tissue sections, with recommended dilutions typically between 1:50 and 1:300 .

• Enzyme-Linked Immunosorbent Assay (ELISA): Provides quantitative measurement of phosphorylated MAP3K5 levels in complex samples, with recommended dilutions around 1:20000 .

These applications enable researchers to examine the phosphorylation status of MAP3K5 at Ser83 across different experimental conditions, cell types, and disease models, facilitating investigation of stress response pathways, apoptosis regulation, and immune signaling mechanisms.

What considerations are important for Western blot detection of phosphorylated MAP3K5?

Successful Western blot detection of phosphorylated MAP3K5 at Ser83 requires careful attention to several methodological aspects:

• Sample Preparation: Rapid and appropriate sample handling is crucial to preserve phosphorylation status. Samples should be collected quickly and lysed in buffers containing phosphatase inhibitors to prevent dephosphorylation during processing .

• Expected Molecular Weight: MAP3K5/ASK1 has a calculated molecular weight of approximately 155 kDa, which should be considered when analyzing Western blot results .

• Antibody Selection and Validation: It is essential to use antibodies that specifically recognize the phosphorylated form of MAP3K5 at Ser83. According to manufacturer information, these antibodies detect endogenous levels of MAP3K5 only when phosphorylated at Ser83 .

• Controls: Proper experimental design should include positive controls (such as lysates from cells treated with known inducers of Ser83 phosphorylation) and negative controls (such as phosphatase-treated samples or samples from cells with MAP3K5 knockdown) .

• Quantification: For quantitative analysis, it is recommended to normalize phospho-MAP3K5 signal to total MAP3K5 levels to account for variations in protein expression between samples. This requires parallel blotting with an antibody recognizing total MAP3K5 regardless of phosphorylation status .

How can immunohistochemistry protocols be optimized for phospho-epitope detection?

Optimizing immunohistochemistry protocols for phospho-MAP3K5 (Ser83) detection requires addressing several challenges unique to phospho-epitopes:

• Tissue Fixation and Processing: Phospho-epitopes are often sensitive to fixation conditions. Formalin/PFA-fixed paraffin-embedded sections are commonly used, but fixation time should be carefully controlled to preserve phosphorylation while achieving adequate tissue morphology .

• Antigen Retrieval: Heat-induced epitope retrieval methods are typically necessary to unmask phospho-epitopes that may become obscured during fixation. The specific pH and buffer composition should be optimized for phospho-MAP3K5 detection .

• Antibody Dilution: Manufacturers recommend dilutions ranging from 1:50 to 1:300 for IHC applications. Optimization experiments testing multiple dilutions are advisable for each specific tissue type and experimental condition .

• Signal Amplification: Secondary detection systems may need enhancement for optimal visualization of phospho-signals, which can be relatively low abundance compared to total protein levels.

• Validation: Parallel staining of serial sections with antibodies against total MAP3K5 helps confirm specificity and provides context for interpreting phosphorylation patterns. Additional validation approaches include the use of tissues known to exhibit differential phosphorylation of MAP3K5 at Ser83 .

How should experiments be designed to study MAP3K5/ASK1 phosphorylation dynamics?

Robust experimental design for studying MAP3K5/ASK1 phosphorylation dynamics should incorporate the following elements:

• Temporal Analysis: Include multiple time points after stimulus application to capture the kinetics of phosphorylation and dephosphorylation events. MAP3K5/ASK1 activation often shows complex temporal patterns that can be missed with single time point measurements .

• Dose-Response Relationships: Test multiple concentrations of stimuli (e.g., oxidative stress inducers, inflammatory cytokines) to determine threshold levels required for Ser83 phosphorylation and to characterize dose-dependent effects .

• Multiple Detection Methods: Combine complementary approaches (e.g., Western blot, IHC, ELISA) to strengthen findings and overcome limitations of individual techniques .

• Pathway Perturbation: Include experiments with specific inhibitors or activators of upstream regulators to establish causal relationships in signaling pathways leading to MAP3K5 Ser83 phosphorylation .

• Genetic Approaches: Consider using gene editing techniques (CRISPR/Cas9) to generate Ser83 mutants (e.g., S83A to prevent phosphorylation or S83D/E to mimic constitutive phosphorylation) for functional studies .

• Parallel Assessment of Multiple Phosphorylation Sites: Simultaneously monitor phosphorylation at different sites (e.g., Ser83 and Ser966) to understand their interdependence and relative contributions to MAP3K5/ASK1 regulation .

What positive and negative controls are essential when investigating MAP3K5 phosphorylation?

Rigorous control experiments are critical for reliable investigation of MAP3K5 phosphorylation:

Positive Controls:

• Known Inducers: Treatments with established inducers of MAP3K5 Ser83 phosphorylation, such as specific oxidative stress conditions or TNF-α, can serve as positive controls .

• Phospho-Mimetic Mutants: Cells expressing phospho-mimetic Ser83 variants can provide reference signals for phosphorylated states.

• Validated Cell Lines: Human cell lines like 293T cells that have been verified to exhibit detectable phospho-MAP3K5 (Ser83) signals under appropriate conditions .

Negative Controls:

• Phosphatase Treatment: Sample aliquots treated with phosphatases to remove phosphorylation can demonstrate signal specificity .

• Blocking Peptides: Competition assays using the phospho-peptide immunogen can confirm antibody specificity .

• Phospho-Deficient Mutants: Cells expressing S83A mutants that cannot be phosphorylated at this site provide important negative controls .

• MAP3K5/ASK1 Knockdown/Knockout: Cells with reduced or eliminated MAP3K5 expression help validate signal specificity and establish background levels .

How can researchers validate the specificity of phospho-MAP3K5 (Ser83) antibody detection?

Validating antibody specificity is paramount for reliable phosphorylation studies. Multiple complementary approaches should be employed:

• Antibody Pair Analysis: Using antibody pairs that recognize total MAP3K5 and phospho-MAP3K5 (Ser83) in parallel provides internal validation. The phospho-specific signal should represent a subset of the total MAP3K5 signal .

• Peptide Competition: Pre-incubating the antibody with synthetic phosphopeptides corresponding to the Ser83 region should abolish specific signals if the antibody is truly phospho-specific .

• Mutagenesis Studies: Comparing signals between wild-type MAP3K5 and S83A mutants provides definitive validation of phospho-specificity .

• Phosphatase Controls: Treating samples with lambda phosphatase should eliminate phospho-specific signals while leaving total MAP3K5 signals intact .

• Multiple Antibody Comparison: Using different phospho-MAP3K5 (Ser83) antibodies from different manufacturers or production lots can help confirm findings and identify potential antibody-specific artifacts .

What are common challenges in detecting phospho-MAP3K5 (Ser83) and their solutions?

Researchers frequently encounter several technical challenges when detecting phospho-MAP3K5 (Ser83):

How should researchers interpret changes in MAP3K5 Ser83 phosphorylation in the context of cellular signaling pathways?

Interpreting changes in MAP3K5 Ser83 phosphorylation requires consideration of the broader signaling context:

• Relative Quantification: Express phospho-MAP3K5 (Ser83) levels relative to total MAP3K5 to account for changes in protein expression that might confound phosphorylation data .

• Temporal Context: Consider the kinetics of phosphorylation changes, as transient versus sustained phosphorylation may have different functional implications for downstream signaling .

• Multi-Site Phosphorylation: Interpret Ser83 phosphorylation in conjunction with other phosphorylation sites (e.g., Ser966) to develop a comprehensive understanding of MAP3K5 regulation .

• Downstream Pathway Activation: Correlate Ser83 phosphorylation with the activation status of downstream targets in the MAP kinase cascade (e.g., phosphorylation of MAP2K4/SEK1, MAP2K3/MKK3, MAP2K6/MKK6, and MAP2K7/MKK7) to establish functional relevance .

• Biological Outcomes: Connect phosphorylation changes to relevant cellular outcomes such as apoptosis, stress responses, or immune function to establish physiological significance .

What factors can lead to false positive or false negative results when detecting phospho-MAP3K5?

Several factors can compromise the reliability of phospho-MAP3K5 detection:

Potential False Positives:

• Antibody cross-reactivity with similar phospho-epitopes on other proteins

• Inadequate blocking leading to non-specific binding

• Artifactual phosphorylation during sample handling due to stress-induced kinase activation

• Signal amplification methods that enhance background along with specific signals

Potential False Negatives:

• Rapid dephosphorylation during sample preparation if phosphatase inhibitors are inadequate

• Epitope masking due to protein-protein interactions or conformational changes

• Insufficient sensitivity of detection methods for low-abundance phosphorylation events

• Improper storage conditions leading to antibody degradation (avoid freeze-thaw cycles)

To minimize these risks, researchers should implement rigorous controls, validate findings using multiple methodologies, and carefully optimize experimental conditions based on the specific requirements of phospho-MAP3K5 (Ser83) detection .

How can phospho-MAP3K5 (Ser83) analysis contribute to understanding disease mechanisms?

Phosphorylation of MAP3K5/ASK1 at Ser83 plays crucial roles in various pathological processes, making it a valuable target for disease-related research:

• Neurodegenerative Disorders: MAP3K5/ASK1 activation is implicated in neuronal cell death pathways relevant to conditions like Alzheimer's and Parkinson's diseases. Monitoring Ser83 phosphorylation can provide insights into stress-response mechanisms in neurodegeneration .

• Cancer Biology: Altered regulation of apoptotic pathways involving MAP3K5/ASK1 contributes to cancer development and therapy resistance. Phospho-MAP3K5 analysis in tumor samples can reveal disruptions in cell death signaling pathways .

• Inflammatory Diseases: Given MAP3K5's role in innate immune responses, analyzing its phosphorylation status in inflammatory conditions can illuminate aberrant immune signaling mechanisms .

• Cardiac Pathologies: MAP3K5/ASK1 mediates stress responses in cardiomyocytes. Phospho-specific analysis can help understand cellular adaptations in heart failure and ischemia-reperfusion injury .

Researchers can apply phospho-MAP3K5 (Ser83) analysis to patient-derived samples, disease models, and therapeutic intervention studies to elucidate pathogenic mechanisms and identify potential targets for therapeutic modulation .

What advanced technologies can enhance phospho-MAP3K5 detection and analysis?

Several cutting-edge approaches can extend the capabilities of traditional phospho-MAP3K5 detection methods:

• Proximity Ligation Assay (PLA): This technique allows visualization of phospho-MAP3K5 in situ with enhanced specificity by detecting proximity between total MAP3K5 and phospho-Ser83 antibodies. PLA-based antibody pairs are commercially available for this purpose .

• Mass Spectrometry-Based Phosphoproteomics: This approach enables unbiased, comprehensive analysis of phosphorylation events on MAP3K5/ASK1 and interacting partners, providing a systems-level view of signaling networks .

• Single-Cell Phospho-Protein Analysis: Techniques like mass cytometry (CyTOF) or single-cell Western blotting allow examination of phospho-MAP3K5 heterogeneity within cell populations, revealing subpopulation-specific signaling dynamics .

• Live-Cell Imaging with Phospho-Sensors: Genetically-encoded fluorescent biosensors designed to report MAP3K5 phosphorylation status enable real-time visualization of signaling dynamics in living cells .

• Computational Modeling: Integration of phosphorylation data into mathematical models can predict system behaviors and generate testable hypotheses about MAP3K5 regulation in complex signaling networks .

How can researchers investigate the interplay between Ser83 phosphorylation and other regulatory mechanisms of MAP3K5/ASK1?

Understanding the complex regulation of MAP3K5/ASK1 requires investigation of multiple regulatory mechanisms and their interactions:

• Multi-Site Phosphorylation Analysis: Simultaneous examination of phosphorylation at Ser83 and other sites (e.g., Ser966) can reveal synergistic or antagonistic relationships between different phosphorylation events .

• Protein-Protein Interaction Studies: Investigating how Ser83 phosphorylation affects MAP3K5 binding to regulatory partners (e.g., 14-3-3 proteins, thioredoxin) provides insights into phosphorylation-dependent protein complex formation .

• Post-Translational Modification Crosstalk: Analyzing interactions between phosphorylation and other modifications (e.g., oxidation, ubiquitination) can uncover complex regulatory networks controlling MAP3K5 activity .

• Structural Biology Approaches: X-ray crystallography or cryo-EM studies of MAP3K5 in different phosphorylation states can reveal conformational changes induced by Ser83 phosphorylation that affect kinase activity .

• Genetic Approaches: Creating cell lines with phospho-mimetic or phospho-deficient mutations at Ser83 and other regulatory sites allows dissection of site-specific functions in cellular responses to various stimuli .