Phospho-MAP3K7 (T187) Antibody

What is MAP3K7 and what is the significance of T187 phosphorylation?

MAP3K7, also known as Transforming growth factor-beta-activated kinase 1 (TAK1), is a serine/threonine kinase that functions as an essential component of the MAP kinase signal transduction pathway. It plays a critical role in mediating cellular responses to environmental changes and transducing signals from various receptors including TGF-beta and IL-1 receptors . The protein has a calculated molecular weight of approximately 67 kDa .

Phosphorylation at threonine 187 (T187) is one of several regulatory phosphorylation sites on MAP3K7 that modulates its kinase activity. This specific phosphorylation event is crucial for the activation of downstream pathways including JNK, p38 MAPK, and NF-κB signaling cascades that control diverse cellular processes such as inflammation, immune responses, cell survival, and apoptosis . T187 phosphorylation occurs in response to various stimuli and serves as a biomarker for MAP3K7 activation status in experimental systems.

What applications are supported for Phospho-MAP3K7(T187) antibodies?

Phospho-MAP3K7(T187) antibodies support multiple experimental applications that enable researchers to study the activation state of MAP3K7 in various biological contexts:

These applications allow researchers to detect and quantify the phosphorylation status of MAP3K7 at T187 in cell and tissue samples, enabling investigations into signaling pathway activation under various experimental conditions .

How specific are Phospho-MAP3K7(T187) antibodies?

Commercially available Phospho-MAP3K7(T187) antibodies are typically generated using synthetic phosphopeptides corresponding to amino acid residues surrounding T187 of human MAP3K7. These antibodies undergo rigorous validation to ensure specificity for the phosphorylated form of the protein .

Specificity validation typically includes:

• Dot blot analysis comparing reactivity with phosphorylated versus non-phosphorylated peptides

• Western blot analysis with phosphatase-treated versus untreated samples

• Peptide competition assays to confirm epitope specificity

• Cross-reactivity testing against other phosphoproteins

What are the optimal storage and handling conditions for Phospho-MAP3K7(T187) antibodies?

Proper storage and handling of Phospho-MAP3K7(T187) antibodies are critical for maintaining their specificity and reactivity. Based on manufacturer recommendations:

For short-term storage (up to 1 month):

• Store at 2-8°C in the original container

• Avoid contamination with microorganisms

• Protect from exposure to light

For long-term storage (up to 1 year or longer):

• Store at -20°C in small aliquots (10-50 μL)

• Avoid repeated freeze-thaw cycles (no more than 2-3)

• Add glycerol (final concentration 50%) to prevent freezing damage

Most commercial preparations come in PBS buffer containing preservatives such as 0.09% sodium azide and stabilizers like 0.5% BSA . When handling these antibodies, researchers should wear appropriate personal protective equipment due to the presence of sodium azide, which is toxic and can form explosive compounds with heavy metals in plumbing systems .

How can Western blot protocols be optimized for Phospho-MAP3K7(T187) detection?

Optimizing Western blot protocols for Phospho-MAP3K7(T187) detection requires attention to several critical factors:

• Sample preparation:

  • Lyse cells in buffer containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Process samples quickly and maintain at 4°C to prevent dephosphorylation

  • Include positive controls (cells treated with TGF-β or IL-1)

• Gel electrophoresis and transfer:

  • Use 8-10% SDS-PAGE gels for optimal resolution of the 67 kDa MAP3K7 protein

  • Transfer to PVDF membranes (rather than nitrocellulose) for better retention of phosphoproteins

  • Use wet transfer systems at low voltage (30V) overnight at 4°C for improved transfer efficiency

• Blocking and antibody incubation:

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

  • Incubate with Phospho-MAP3K7(T187) antibody at 1:500-1:1000 dilution overnight at 4°C

  • Use TBS-based buffers instead of PBS to avoid phosphate interference

• Detection optimization:

  • Use enhanced chemiluminescence systems with extended exposure times if signal is weak

  • Consider signal amplification systems for low abundance targets

  • Include molecular weight markers to confirm band identity at approximately 67 kDa

What species cross-reactivity can be expected with Phospho-MAP3K7(T187) antibodies?

Based on the available data, Phospho-MAP3K7(T187) antibodies demonstrate the following species cross-reactivity:

The cross-reactivity is based on the conservation of the amino acid sequence surrounding the T187 phosphorylation site across these species . When working with species other than human, preliminary validation experiments are recommended to confirm antibody performance. Researchers should note that even when the sequence is conserved, differences in protein expression levels, post-translational modifications, or tissue-specific isoforms may affect detection sensitivity.

What signaling pathways involve MAP3K7 phosphorylation at T187?

MAP3K7 (TAK1) phosphorylation at T187 is a critical regulatory event in multiple signaling pathways:

• TGF-β/BMP signaling pathway:

  • TGF-β binding to its receptor activates MAP3K7 through interaction with TRAF6

  • T187 phosphorylation enhances MAP3K7 kinase activity

  • Activated MAP3K7 phosphorylates downstream targets including MAPK kinases

• Inflammatory signaling:

  • IL-1 and TNF-α stimulation leads to MAP3K7 activation

  • Phosphorylated MAP3K7 forms a complex with MAP3K7P1/TAB1 and MAP3K7P2/TAB2

  • This complex is essential for activation of NF-κB, which controls expression of inflammatory genes

• Stress response pathway:

  • Environmental stresses trigger MAP3K7 T187 phosphorylation

  • Activated MAP3K7 phosphorylates and activates MAPK8/JNK and MAP2K4/MKK4

  • These kinases mediate cellular adaptation to stress conditions

The phosphorylation at T187 serves as a molecular switch that changes MAP3K7 conformation, enhancing its catalytic activity and enabling interaction with downstream substrates. This phosphorylation event is therefore a critical biomarker for the activation status of these pathways in experimental systems.

How does T187 phosphorylation compare to other phosphorylation sites on MAP3K7?

MAP3K7 contains several phosphorylation sites that regulate its activity through different mechanisms:

T187 phosphorylation is considered one of the most critical regulatory events for MAP3K7 activation. Unlike some other phosphorylation sites that may have context-dependent effects, T187 phosphorylation consistently correlates with increased kinase activity . When designing experiments to study MAP3K7 regulation, researchers should consider the interdependence of these phosphorylation events and potentially examine multiple sites simultaneously to gain a comprehensive understanding of the activation state.

What experimental controls are crucial when studying MAP3K7 T187 phosphorylation?

When studying MAP3K7 T187 phosphorylation, several critical controls should be included:

• Positive controls:

  • Cells treated with known MAP3K7 activators (TGF-β, IL-1β, or TNF-α)

  • Recombinant phosphorylated MAP3K7 peptide (when available)

  • Lysates from cells overexpressing constitutively active MAP3K7

• Negative controls:

  • Untreated/unstimulated cells

  • Cells treated with MAP3K7 inhibitors (e.g., 5Z-7-Oxozeaenol)

  • Phosphatase-treated samples to remove phosphorylation

  • MAP3K7 knockdown or knockout cells

• Antibody controls:

  • Non-phospho-specific MAP3K7 antibody to assess total protein levels

  • Peptide competition assays using phosphorylated vs. non-phosphorylated peptides

  • Secondary antibody-only controls to detect non-specific binding

Including these controls helps validate findings and ensures that any observed changes in T187 phosphorylation are specific and biologically relevant. Additionally, time-course experiments can provide insights into the dynamics of phosphorylation events following stimulation.

How can I troubleshoot non-specific binding when using Phospho-MAP3K7(T187) antibody?

Non-specific binding is a common challenge when working with phospho-specific antibodies. To troubleshoot this issue with Phospho-MAP3K7(T187) antibody:

• Optimize blocking conditions:

  • Increase blocking time (2-3 hours at room temperature)

  • Test different blocking agents (5% BSA, 5% non-fat dry milk, commercial blocking buffers)

  • Add 0.1-0.3% Tween-20 to reduce background

• Adjust antibody conditions:

  • Titrate antibody concentration (test dilutions from 1:250 to 1:2000)

  • Reduce incubation temperature (4°C overnight instead of room temperature)

  • Perform more stringent washing steps (5-6 washes of 10 minutes each)

• Validate specificity:

  • Perform peptide competition assays with phosphorylated peptide

  • Compare reactivity in phosphatase-treated vs. untreated samples

  • Test the antibody on MAP3K7 knockout/knockdown samples

• Modify sample preparation:

  • Ensure complete cell lysis and protein denaturation

  • Add phosphatase inhibitors immediately during lysis

  • Centrifuge lysates at high speed to remove insoluble material

For dot blot applications specifically, antibody specificity can be directly assessed by comparing reactivity with phospho-peptide versus non-phospho-peptide targets at varying antibody concentrations (0.1-1.0 μg/ml) .

What methods can be used to induce MAP3K7 T187 phosphorylation in vitro?

Several methods can reliably induce MAP3K7 T187 phosphorylation in experimental systems:

• Cytokine stimulation:

  • TGF-β1: 2-10 ng/ml for 15-30 minutes

  • IL-1β: 10 ng/ml for 5-15 minutes

  • TNF-α: 10-20 ng/ml for 10-30 minutes

• Receptor activation:

  • Toll-like receptor agonists (LPS: 100 ng/ml for 30 minutes)

  • BMP receptor activation (BMP2: 100 ng/ml for 30-60 minutes)

• Stress induction:

  • Osmotic stress (sorbitol: 0.4-0.6 M for 30 minutes)

  • Oxidative stress (H₂O₂: 100-500 μM for 15-30 minutes)

  • UV irradiation (40 J/m² followed by 30-minute recovery)

• Chemical inducers:

  • Anisomycin: 10 μg/ml for 30 minutes

  • Phorbol esters (PMA): 100 nM for 30-60 minutes

Cell type-specific differences in response magnitude and timing should be considered when designing experiments. Preliminary time-course and dose-response studies are recommended to determine optimal conditions for each experimental system. For maximal phosphorylation, pre-treatment with phosphatase inhibitors like okadaic acid (100 nM) can enhance detection of the phosphorylated form.

How is MAP3K7 T187 phosphorylation implicated in disease models?

MAP3K7 T187 phosphorylation has been implicated in several disease contexts, making it an important target for translational research:

• Inflammatory disorders:

  • Aberrant MAP3K7 activation contributes to chronic inflammation

  • T187 phosphorylation is elevated in arthritis models

  • Inhibition of MAP3K7 activation shows therapeutic potential in inflammatory bowel disease

• Cancer biology:

  • Dysregulated MAP3K7 signaling affects tumor cell survival and proliferation

  • Altered T187 phosphorylation status has been observed in certain cancer types

  • MAP3K7 modulation influences chemotherapy response

• Cardiovascular disease:

  • MAP3K7 activation via T187 phosphorylation mediates cardiac hypertrophy

  • Stress-induced MAP3K7 phosphorylation contributes to cardiomyocyte death

  • Inhibition of MAP3K7 shows cardioprotective effects in ischemia-reperfusion models

• Neurodegenerative disorders:

  • Neuroinflammatory processes involve MAP3K7 activation

  • T187 phosphorylation is altered in models of Alzheimer's and Parkinson's disease

  • Targeting MAP3K7 signaling shows neuroprotective potential

When designing experiments to study MAP3K7 T187 phosphorylation in disease models, researchers should consider tissue-specific expression patterns, the temporal dynamics of phosphorylation, and the integration with other signaling pathways relevant to the specific disease context.

What novel methodological approaches are emerging for studying MAP3K7 phosphorylation?

Several cutting-edge approaches are enhancing our ability to study MAP3K7 T187 phosphorylation:

• Phosphoproteomics:

  • Mass spectrometry-based quantification of phosphorylation stoichiometry

  • Multiplexed analysis of multiple phosphorylation sites simultaneously

  • Integration with global phosphoproteome analysis for pathway context

• Live-cell imaging:

  • Genetically encoded FRET-based biosensors for MAP3K7 activation

  • Phospho-specific intrabodies for real-time visualization

  • Optogenetic tools to spatiotemporally control MAP3K7 activation

• Single-cell analysis:

  • Phospho-flow cytometry for heterogeneity assessment

  • Single-cell Western blotting for enhanced sensitivity

  • Integration with transcriptomics for multi-omic analysis

• Structural biology approaches:

  • Cryo-EM studies of MAP3K7 conformational changes upon T187 phosphorylation

  • Hydrogen-deuterium exchange mass spectrometry to map structural dynamics

  • Computational modeling of phosphorylation effects on protein-protein interactions

These emerging methodologies offer unprecedented resolution for studying MAP3K7 phosphorylation events in complex biological systems. Combining multiple approaches can provide complementary insights into the regulation and function of MAP3K7 T187 phosphorylation in different experimental contexts.