Phospho-MAP2K4 (Thr261) Antibody

What is Phospho-MAP2K4 (Thr261) Antibody and how does it function in research?

Phospho-MAP2K4 (Thr261) Antibody is a specialized research reagent designed to detect the dual specificity mitogen-activated protein kinase kinase 4 (MAP2K4, also known as SEK1/MKK4) only when phosphorylated at threonine 261. The antibody specifically recognizes this phosphorylated form, making it an essential tool for monitoring MAP2K4 activation states in signaling pathway research . These antibodies are typically developed in rabbits as polyclonal antibodies through immunization with synthetic phosphopeptides corresponding to the region surrounding Thr261 of human MAP2K4 .

The function of these antibodies is to provide researchers with the ability to monitor the activation state of MAP2K4, which serves as an essential component of the stress-activated protein kinase/c-Jun N-terminal kinase (SAP/JNK) signaling pathway. Since MAP2K4 activation requires phosphorylation at both Ser257 and Thr261 by upstream MAP kinase kinase kinases (MAP3Ks), these antibodies allow researchers to track specific phosphorylation events in complex signaling cascades .

What are the technical specifications and validated applications for Phospho-MAP2K4 (Thr261) Antibody?

Phospho-MAP2K4 (Thr261) antibodies are available with several consistent technical specifications that researchers should consider when planning experiments:

The antibody has been validated for multiple research applications with the following typical dilution ranges :

It's important to note that these antibodies are strictly for research use only (RUO) and must not be used in diagnostic or therapeutic applications .

What biological information can be derived from experiments using Phospho-MAP2K4 (Thr261) Antibody?

Experiments utilizing Phospho-MAP2K4 (Thr261) antibodies can reveal critical biological information about MAP kinase signaling pathways, including:

• Activation status of the stress-responsive JNK pathway in various biological contexts

• Temporal dynamics of MAP2K4 activation following exposure to stress stimuli

• Cell type-specific differences in MAP2K4 phosphorylation

• Spatial distribution of activated MAP2K4 within cells (nuclear versus cytoplasmic localization)

• Relationships between MAP2K4 activation and downstream effectors like JNK and c-Jun

The antibody can detect endogenous levels of phosphorylated MAP2K4 at approximately 44 kDa in Western blots . In immunofluorescence studies, phosphorylated MAP2K4 typically shows localization to both nuclear and cytoplasmic compartments, reflecting its dynamic role in signal transduction .

How should I optimize Western blot protocols when using Phospho-MAP2K4 (Thr261) Antibody?

When designing Western blot experiments with Phospho-MAP2K4 (Thr261) antibody, several methodological considerations can maximize sensitivity and specificity:

• Sample preparation protocol:

  • Include phosphatase inhibitor cocktails in all lysis buffers

  • Process samples quickly and maintain cold temperature throughout

  • Use freshly prepared samples whenever possible

  • Consider using phosphoprotein enrichment methods for low-abundance signals

• Optimized blotting conditions:

  • Use PVDF membranes rather than nitrocellulose for better retention of phosphoproteins

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

  • Incubate with primary antibody (1:1000 dilution) overnight at 4°C

  • Use HRP-conjugated anti-rabbit IgG secondary antibody with validated specificity

• Experimental controls and validation:

  • Include phosphatase-treated lysates as negative controls

  • Use lysates from cells treated with 300 mM sorbitol (30 minutes) or 10 ng/mL IL-1β (20 minutes) as positive controls

  • Run parallel blots for total MAP2K4 to normalize phospho-specific signals

  • Expect a specific band at approximately 44 kDa

This approach has been validated in published research showing detection of phosphorylated MAP2K4 in PC-12 rat adrenal pheochromocytoma and HepG2 human hepatocellular carcinoma cell lines following stress induction .

What are methodological considerations for immunofluorescence experiments with Phospho-MAP2K4 (Thr261) Antibody?

For immunofluorescence applications, consider this methodological approach to achieve optimal results:

• Cell preparation:

  • Grow cells on coated coverslips to appropriate confluence (60-80%)

  • Apply appropriate stimuli to induce MAP2K4 phosphorylation

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

• Antibody staining:

  • Block with 5% normal goat serum for 1 hour at room temperature

  • Apply primary antibody at 1:50-1:200 dilution in blocking buffer

  • Incubate overnight at 4°C in a humidified chamber

  • Wash extensively with PBS (3-5 times, 5 minutes each)

  • Apply fluorophore-conjugated anti-rabbit secondary antibody (e.g., NorthernLights 557-conjugated)

  • Counterstain nuclei with DAPI

• Imaging and analysis:

  • Use confocal microscopy for subcellular localization studies

  • Capture z-stacks for three-dimensional analysis if necessary

  • Quantify nuclear versus cytoplasmic signal distribution

  • Compare staining patterns between treatment conditions

Published research has demonstrated that this approach can successfully detect phosphorylated MAP2K4 in HeLa cells, with specific staining localized to both nuclei and cytoplasm . The specificity of this staining can be validated using phosphatase treatment, which eliminates the signal in negative control cells .

How can I validate the specificity of phospho-signal detection in my experiments?

• Phosphatase treatment controls:

  • Divide your sample into two portions

  • Treat one portion with calf intestinal phosphatase (CIP)

  • Process both samples identically for Western blot or immunostaining

  • A true phospho-specific signal should be eliminated or significantly reduced in the phosphatase-treated sample

• Stimulus-response validation:

  • Compare untreated samples with samples exposed to known activators

  • For MAP2K4, established activators include:

    • Osmotic stress (300 mM sorbitol, 30 minutes)

    • Inflammatory cytokines (10 ng/mL IL-1β, 20 minutes)

    • UV irradiation

  • A genuine phospho-specific signal should increase proportionally with stimulus intensity

• Genetic validation approaches:

  • Use MAP2K4 knockdown or knockout models

  • Compare antibody signal in wild-type versus MAP2K4-deficient samples

  • The specific phospho-signal should be absent or significantly reduced in knockout conditions

• Multiple detection methods:

  • Confirm results using different techniques (Western blot, immunofluorescence, ELISA)

  • Each method should show consistent patterns of MAP2K4 phosphorylation

This validation approach has been demonstrated in research showing that phosphorylated MAP2K4 signal is absent in HeLa cells treated with CIP, confirming the phospho-specificity of the antibody .

How can I distinguish between MAP2K4 phosphorylation at different sites (Ser257 vs. Thr261)?

Differentiating between phosphorylation at different sites on MAP2K4 requires careful methodological considerations:

• Site-specific antibody selection:

  • Use antibodies that specifically recognize phospho-Thr261

  • Compare with antibodies specific for phospho-Ser257

  • Consider dual-phospho antibodies that recognize both sites simultaneously (e.g., phospho-S257/T261)

• Temporal dynamics analysis:

  • Conduct time-course experiments after stimulation

  • Different sites may show distinct kinetics of phosphorylation and dephosphorylation

  • Plot relative phosphorylation levels over time for each site

• Kinase inhibitor approach:

  • Apply inhibitors of different upstream MAP3Ks

  • Determine which inhibitors differentially affect phosphorylation at Ser257 versus Thr261

  • This approach can reveal kinase-specific preferences for different phosphorylation sites

• Mutational analysis:

  • Create phosphomimetic (S257D, T261D) or phosphodeficient (S257A, T261A) mutants

  • Compare signaling outcomes with single versus double mutants

  • This approach can distinguish the functional importance of each phosphorylation site

Research indicates that both Ser257 and Thr261 phosphorylation are required for full activation of MAP2K4, but they may be regulated by different upstream kinases in a context-dependent manner .

What controls are essential for interpreting experiments with Phospho-MAP2K4 (Thr261) Antibody?

Proper experimental controls are critical for accurate interpretation of results with phospho-specific antibodies:

Research has shown that treatment with KRAS inhibitors like sotorasib or MEK inhibitors like trametinib induces MAP2K4-JNK-JUN pathway activation that peaks at 48-72 hours, emphasizing the importance of appropriate time points for different experimental contexts .

How should I interpret contradictory results between phospho-MAP2K4 levels and downstream pathway activation?

When faced with discrepancies between MAP2K4 phosphorylation and downstream signaling, consider these analytical approaches:

• Temporal disconnect analysis:

  • Phosphorylation events often show different temporal dynamics

  • MAP2K4 phosphorylation may precede JNK activation

  • Plot time courses of phosphorylation for MAP2K4, JNK, and c-Jun to identify temporal relationships

• Signal amplitude assessment:

  • Threshold effects may exist where minimal MAP2K4 activation is sufficient for maximal downstream signaling

  • Quantify the relationship between phospho-MAP2K4 levels and phospho-JNK/c-Jun levels

  • Consider creating dose-response curves with varying stimulus intensities

• Pathway crosstalk evaluation:

  • JNK can be activated by both MAP2K4 and MAP2K7

  • MAP2K7 may compensate for reduced MAP2K4 activity

  • Investigate MAP2K7 phosphorylation status in parallel

  • Research indicates MAP2K4 preferentially phosphorylates the tyrosine residue in JNK, while MAP2K7 targets the threonine residue

• Subcellular compartmentalization:

  • Analyze nuclear versus cytoplasmic fractions separately

  • Signal propagation may occur in specific cellular compartments

  • Phosphorylated MAP2K4 has been observed in both nuclear and cytoplasmic locations

Resolving such contradictions often reveals important regulatory mechanisms in the signaling pathway that would otherwise remain undiscovered.

How can Phospho-MAP2K4 (Thr261) Antibody be used to investigate cancer resistance mechanisms?

Recent research has revealed critical roles for MAP2K4 phosphorylation in cancer therapy resistance that can be investigated using Phospho-MAP2K4 (Thr261) antibodies:

• MAP2K4-mediated feedback activation in KRAS-mutant cancers:

  • KRAS G12C inhibitors (sotorasib) and MEK inhibitors (trametinib) induce a MAP2K4-dependent feedback mechanism in lung and colon cancer models

  • This feedback activates JUN, which upregulates ERBB2 and ERBB3 receptor tyrosine kinases

  • The upregulated RTK signaling reactivates KRAS and downstream MAPK pathway, limiting drug efficacy

• Experimental protocols to study this mechanism:

  • Treat cancer cell lines with KRAS inhibitors for 48-72 hours

  • Monitor phospho-MAP2K4, phospho-JUN, total/phospho-ERBB2, and total/phospho-ERBB3 levels

  • Compare wild-type cells with MAP2K4 knockout cells

  • Assess downstream pathway activity through phospho-ERK levels

• Combination therapy investigation:

  • Co-treat cells with KRAS inhibitors and MAP2K4 inhibitors (e.g., HRX-0233)

  • Monitor suppression of phospho-JUN upregulation

  • Assess ERBB2/3 expression and phosphorylation

  • Measure sustained inhibition of phospho-ERK signaling

Research has demonstrated that MAP2K4 loss or inhibition enhances sensitivity to KRAS pathway inhibitors in multiple cancer models, providing a rational basis for combination therapy approaches targeting both KRAS and MAP2K4 .

What methodological approaches can reveal the role of MAP2K4 phosphorylation in cellular stress responses?

To investigate MAP2K4's role in stress responses, consider these advanced methodological strategies:

• Stress-specific phosphorylation dynamics:

  • Apply different stressors (oxidative, genotoxic, inflammatory, metabolic)

  • Monitor phospho-MAP2K4 (Thr261) levels at multiple time points (5min to 24h)

  • Compare with activation of other stress-responsive pathways (p38, NF-κB)

  • Correlate MAP2K4 phosphorylation with cellular outcomes (apoptosis, senescence, adaptation)

• Upstream kinase identification:

  • Apply specific inhibitors of MAP3Ks (MEKK1/2/3, MLK2/3, ASK1/2)

  • Use siRNA/shRNA to knockdown specific MAP3Ks

  • Measure phospho-MAP2K4 (Thr261) levels after each intervention

  • This approach can identify which kinases phosphorylate MAP2K4 under specific stress conditions

• Scaffolding protein interactions:

  • Perform co-immunoprecipitation with phospho-MAP2K4 (Thr261) antibody

  • Identify associated proteins by mass spectrometry

  • Confirm interactions by reciprocal immunoprecipitation

  • Map interaction dynamics during stress response time course

• Functional consequences of phosphorylation:

  • Compare wild-type MAP2K4 with phosphomimetic (T261D) and phosphodeficient (T261A) mutants

  • Assess effects on downstream JNK activation, c-Jun phosphorylation, and transcriptional responses

  • Measure cellular outcomes (proliferation, survival, migration) with each MAP2K4 variant

These approaches can provide comprehensive insights into how MAP2K4 phosphorylation regulates stress-responsive signaling networks in normal and pathological states.

How does MAP2K4 phosphorylation status relate to therapeutic response in cancer models?

The relationship between MAP2K4 phosphorylation and therapeutic response can be investigated using these advanced research protocols:

• Therapeutic response correlation analysis:

  • Create a panel of cancer cell lines with varying MAP2K4 expression/activity

  • Measure baseline phospho-MAP2K4 (Thr261) levels

  • Assess sensitivity to targeted therapies (KRAS inhibitors, MEK inhibitors)

  • Correlate pre-treatment phospho-MAP2K4 levels with drug sensitivity

• Dynamic biomarker potential:

  • Collect sequential samples during treatment (pre-treatment, 6h, 24h, 48h, 72h)

  • Monitor phospho-MAP2K4 (Thr261) levels throughout treatment

  • Identify whether early changes in phospho-MAP2K4 predict later therapeutic response

  • Research shows peak activation of the MAP2K4-JNK-JUN pathway occurs 48-72h after treatment initiation

• Combination therapy development:

  • Design treatment matrices combining MAP2K4 inhibitors with various targeted therapies

  • Measure phospho-MAP2K4 (Thr261) suppression by each combination

  • Correlate pathway inhibition with anti-tumor effects

  • Identify synergistic drug combinations

• In vivo validation models:

  • Establish patient-derived xenograft models

  • Administer single-agent and combination therapies

  • Collect tumor biopsies at multiple time points

  • Measure phospho-MAP2K4 (Thr261) levels by immunohistochemistry

  • Correlate changes in phosphorylation with tumor response

Research has established that MAP2K4 inhibition synergizes with KRAS pathway inhibitors in preclinical models, providing rationale for developing MAP2K4 phosphorylation as both a biomarker and therapeutic target .

What strategies can resolve inconsistent phospho-MAP2K4 detection in Western blot experiments?

When encountering difficulties with phospho-MAP2K4 detection, implement these methodological remedies:

• Sample preparation optimization:

  • Use fresh phosphatase inhibitor cocktails in all buffers

  • Process samples rapidly at 4°C to preserve phosphorylation

  • Consider protein extraction methods specifically designed for phosphoproteins

  • Prevent sample overheating during sonication or processing

• Signal enhancement approaches:

  • Increase sample concentration (load 50-75 μg protein)

  • Use high-sensitivity chemiluminescent substrates

  • Consider phosphoprotein enrichment columns before Western blotting

  • Optimize antibody concentration (try 1:500 dilution if signal is weak)

• Membrane and transfer optimization:

  • Use PVDF membrane instead of nitrocellulose

  • Adjust transfer conditions (lower voltage, longer time)

  • Add SDS (0.1%) to transfer buffer to improve transfer of phosphoproteins

  • Pre-wet membrane in methanol before equilibrating in transfer buffer

• Antibody incubation conditions:

  • Extend primary antibody incubation to overnight at 4°C

  • Use 5% BSA for blocking and antibody dilution (never use milk)

  • Consider gentle agitation during incubation

  • Optimize secondary antibody dilution and incubation time

Published protocols have successfully detected phospho-MAP2K4 using PVDF membranes with 1 μg/mL antibody concentration in cells treated with either sorbitol or IL-1β .

How can I improve phospho-MAP2K4 staining specificity in immunohistochemistry applications?

To enhance specificity and reduce background in immunohistochemistry, consider these methodological refinements:

• Tissue preparation protocol:

  • Fix tissues immediately after collection

  • Limit fixation time to 24 hours

  • Process and embed tissues promptly

  • Cut sections at 3-5 μm thickness for optimal antibody penetration

• Antigen retrieval optimization:

  • Test multiple antigen retrieval methods:

    • Citrate buffer (pH 6.0)

    • EDTA buffer (pH 9.0)

    • Tris-EDTA buffer (pH 8.0)

  • Optimize heating time and temperature

  • Allow gradual cooling to room temperature

• Blocking and antibody incubation:

  • Block with 5-10% normal goat serum

  • Include 0.3% Triton X-100 in blocking solution

  • Use antibody at 1:100 dilution initially

  • Incubate at 4°C overnight in a humidified chamber

  • Extend washing steps (5 × 5 minutes)

• Signal detection system:

  • Use polymer-based detection systems for enhanced sensitivity

  • Consider tyramide signal amplification for very low abundance phosphoproteins

  • Optimize DAB development time (monitor under microscope)

  • Use minimal counterstaining to avoid obscuring specific signal

• Validation controls:

  • Include phosphatase-treated section as negative control

  • Use tissue known to express activated MAP2K4 as positive control

  • Include isotype control antibody on parallel sections

These approaches have been validated in research demonstrating specific detection of phosphorylated MAP2K4 in various tissue types and cell lines .

How can phospho-MAP2K4 antibodies contribute to understanding KRAS inhibitor resistance mechanisms?

Phospho-MAP2K4 (Thr261) antibodies are emerging as critical tools for investigating resistance to KRAS-targeted therapies through these research applications:

• Feedback mechanism characterization:

  • Recent research has identified a MAP2K4-dependent feedback loop activated by KRAS inhibitors

  • This feedback involves JUN activation, which upregulates ERBB2/3 receptors

  • The increased RTK signaling reactivates the MAPK pathway despite KRAS inhibition

  • Phospho-MAP2K4 antibodies enable tracking of this feedback mechanism activation

• Temporal dynamics analysis:

  • Treatment with KRAS G12C inhibitor (sotorasib) activates this feedback pathway

  • Phospho-JUN upregulation is most prominent 48-72 hours after treatment

  • This timing corresponds with therapeutic resistance development

  • Time-course analysis with phospho-MAP2K4 antibodies can map activation kinetics

• Combination therapy development:

  • Small molecule MAP2K4 inhibitor (HRX-0233) prevents feedback activation

  • Measuring phospho-MAP2K4 and downstream phospho-JUN can monitor inhibitor efficacy

  • Combined KRAS and MAP2K4 inhibition provides more sustained suppression of MAPK signaling

  • This approach represents a promising strategy to enhance KRAS inhibitor efficacy

• Predictive biomarker potential:

  • Baseline and treatment-induced changes in phospho-MAP2K4 may predict response

  • Patient biopsies can be analyzed for phospho-MAP2K4 status

  • This information could guide personalized therapy decisions

These approaches are supported by research demonstrating that MAP2K4 loss or inhibition synergizes with KRAS inhibitors in preclinical cancer models .

What methodological advances allow phospho-MAP2K4 detection in limited clinical samples?

Innovative methodological approaches now enable phospho-protein analysis in scarce clinical specimens:

• Microwestern array technology:

  • Miniaturized Western blot format requiring only 0.5-1 μg total protein

  • Allows multiple samples and antibodies on single membrane

  • Can detect phospho-MAP2K4 from limited biopsy material

  • Enables quantitative comparison across multiple patients or conditions

• Reverse phase protein array (RPPA):

  • Immobilizes multiple clinical samples on single slide

  • Probes with phospho-MAP2K4 antibody followed by signal amplification

  • Allows high-throughput screening of phosphorylation status

  • Quantitative readout enables statistical correlation with clinical outcomes

• Single-cell phospho-protein analysis:

  • Flow cytometry with phospho-specific antibodies

  • Mass cytometry (CyTOF) for multi-parameter analysis

  • Single-cell Western blot technologies

  • These approaches reveal heterogeneity in MAP2K4 activation within tumors

• Proximity ligation assay (PLA):

  • Combines antibody specificity with rolling circle amplification

  • Requires two antibodies binding in close proximity

  • Can detect phospho-MAP2K4 in formalin-fixed paraffin-embedded tissues

  • Provides subcellular localization information

These techniques allow researchers to translate findings from cell culture models to clinical specimens, potentially identifying patients who would benefit from therapies targeting the MAP2K4 pathway.

What are the key considerations for designing robust experimental protocols with Phospho-MAP2K4 (Thr261) Antibody?

When designing experiments with Phospho-MAP2K4 (Thr261) antibody, researchers should implement these evidence-based best practices:

• Comprehensive validation:

  • Verify phospho-specificity using phosphatase controls

  • Confirm target specificity using MAP2K4 knockout/knockdown models

  • Include appropriate positive controls (sorbitol or IL-1β treatment)

  • Use multiple detection methods when possible

• Context-appropriate experimental design:

  • Include physiologically relevant stimuli for your research question

  • Design time-course experiments to capture transient phosphorylation

  • Consider pathway cross-talk by monitoring related signaling components

  • Account for cell type-specific differences in MAP2K4 expression and regulation

• Methodological optimization:

  • Preserve phosphorylation status during sample preparation

  • Determine optimal antibody concentration for each application

  • Select appropriate detection methods based on sensitivity requirements

  • Include quantitative analysis whenever possible

• Integrated data interpretation:

  • Consider MAP2K4 phosphorylation in the context of the full pathway

  • Correlate phosphorylation with functional outcomes

  • Acknowledge the limitations of antibody-based detection methods

  • Supplement with orthogonal approaches when available

By adhering to these principles, researchers can generate reliable and reproducible data that advances our understanding of MAP2K4 signaling in normal physiology and disease states.

What future directions are emerging for MAP2K4 phosphorylation research in cancer therapy?

Research into MAP2K4 phosphorylation is revealing promising new directions for cancer therapy development:

• Combination therapy strategies:

  • Co-targeting MAP2K4 with KRAS inhibitors to prevent feedback activation

  • Research shows MAP2K4 inhibition enhances sensitivity to KRAS G12C inhibitors (sotorasib), RAS(ON) multi-inhibitors (RMC-6236), and MEK inhibitors (trametinib)

  • Phospho-MAP2K4 antibodies provide essential tools for monitoring pathway inhibition

• Biomarker development:

  • Using phospho-MAP2K4 status to predict response to targeted therapies

  • Monitoring treatment-induced changes in phospho-MAP2K4 as pharmacodynamic markers

  • Developing companion diagnostics for MAP2K4-targeting therapies

• Novel therapeutic approaches:

  • Designing degraders specifically targeting phosphorylated MAP2K4

  • Exploiting synthetic lethality interactions with MAP2K4-dependent pathways

  • Developing immune approaches that recognize cells with activated MAP2K4

• Expanded application to other cancers:

  • Beyond KRAS-mutant cancers, MAP2K4 signaling may be important in other malignancies

  • Stress-activated pathways are frequently dysregulated across cancer types

  • MAP2K4 inhibition could sensitize tumors to various targeted and conventional therapies