MAPK8 Antibody

What is MAPK8 and what are its alternative names in research literature?

MAPK8 (mitogen-activated protein kinase 8) is a serine/threonine protein kinase also known as JNK1, SAPK1, JNK-46, JNK1A2, SAPK1c, JNK21B1/2, and PRKM8. It belongs to the MAPK superfamily of stress-activated protein kinases and is expressed as multiple isoforms due to differential mRNA splicing, with JNK1 and JNK2 being the predominant forms . MAPK8 is expressed in various tissue types and has a molecular weight of approximately 48.3 kDa, with the canonical human protein consisting of 427 amino acid residues .

What are the primary cellular functions of MAPK8?

MAPK8 functions as a crucial mediator in multiple cellular processes including:

• Signal transduction from cell surface to nucleus

• Cellular proliferation, differentiation, and migration

• Transcription regulation and development

• Apoptosis and cell death pathways

• Response to stress and inflammatory signals

• Insulin resistance and obesity mediation

• Activation in response to misfolded proteins in the endoplasmic reticulum

When activated, MAPK8 phosphorylates various substrates including transcription factors like c-Jun, altering their activities and subcellular localization.

How should researchers choose the appropriate MAPK8 antibody for their specific experimental needs?

When selecting a MAPK8 antibody, researchers should consider:

• Target specificity: Determine whether you need an antibody that specifically recognizes MAPK8/JNK1 or one that recognizes multiple JNK family members (JNK1/2/3)

• Phosphorylation state: Select antibodies that detect total MAPK8 or phosphorylated forms (pT183/pY185)

• Host species compatibility: Ensure minimal cross-reactivity with samples from other species

• Application suitability: Verify antibody validation for your specific application (WB, IHC, IF, ELISA, IP)

• Clone type: Consider whether monoclonal (higher specificity) or polyclonal (broader epitope recognition) is more appropriate

For optimal results, review published literature using your antibody of interest to confirm its reliability in similar experimental contexts.

What validation methods should be used to confirm MAPK8 antibody specificity?

Thorough validation of MAPK8 antibodies should include:

• Western blotting with positive controls: Use cell lysates known to express MAPK8 (e.g., K562 cells) to confirm detection at the expected molecular weight (approximately 44/52 kDa)

• Knockout/knockdown controls: Compare antibody reactivity in wild-type vs. MAPK8-deficient samples

• Phospho-specific validation: For phospho-specific antibodies, compare untreated vs. stimulated samples and/or phosphatase-treated controls

• Cross-reactivity testing: Test against closely related proteins (JNK2/JNK3) to ensure specificity

• Peptide competition assays: Pre-incubate antibody with immunizing peptide to demonstrate signal reduction

Documenting these validation steps is essential for high-quality research publications and reproducibility.

What are the optimal conditions for using MAPK8 antibodies in Western blotting?

For optimal Western blot results with MAPK8 antibodies:

• Sample preparation:

  • Use fresh cell/tissue lysates in RIPA or NP-40 buffer with protease/phosphatase inhibitors

  • For phospho-JNK detection, stimulate cells with appropriate stressors (UV, cytokines, etc.)

• Protein separation:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Load 20-40 μg of total protein per lane

  • Include molecular weight markers spanning 40-60 kDa range

• Transfer and detection:

  • Transfer to PVDF membranes (preferred over nitrocellulose for phospho-epitopes)

  • Block with 5% BSA in TBST (not milk for phospho-antibodies)

  • Primary antibody dilution: typically 1:1000-1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Secondary antibody: 1:5000-1:10000, 1 hour at room temperature

• Expected results:

  • Total JNK1: bands at approximately 44/52 kDa (isoform-dependent)

  • Phospho-JNK: bands at similar molecular weights but only after stimulation

How can MAPK8 antibodies be effectively used in immunohistochemistry and immunofluorescence?

For optimal IHC/IF with MAPK8 antibodies:

• Sample preparation:

  • Fixation: 4% paraformaldehyde (10% formalin) for tissues

  • For IF on cultured cells: 4% PFA for 15 minutes at room temperature

• Antigen retrieval (for fixed tissues):

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • For phospho-epitopes: use EDTA buffer (pH 8.0)

• Blocking and antibody incubation:

  • Block with 5-10% normal serum from secondary antibody host species

  • Primary antibody dilution: typically 1:100-1:500

  • Incubation time: 1-2 hours at room temperature or overnight at 4°C

• Detection systems:

  • For IHC: HRP-conjugated secondary + DAB substrate

  • For IF: fluorophore-conjugated secondary antibody (avoid overlapping wavelengths if co-staining)

• Controls:

  • Include positive control tissues with known MAPK8 expression

  • Include negative controls (secondary antibody only)

  • For phospho-specific antibodies, include samples treated with λ-phosphatase

How is MAPK8 involved in intervertebral disc degeneration, and what methodologies are best for studying this connection?

Recent research identified MAPK8 as a key biomarker in intervertebral disc degeneration (IDD). A 2023 study by Frontiers in Immunology showed:

• MAPK8's role in IDD:

  • Identified as one of six hub genes in autophagy-related gene analysis

  • Validated as differentially expressed in IDD compared to normal tissue

  • Expression correlated with specific immune cell infiltration patterns

• Recommended methodology for studying MAPK8 in IDD:

  • Bioinformatic approaches: Start with DEG analysis followed by MCODE plugin for hub gene identification

  • Animal models: Use needle puncture technique to induce IDD in rats

  • Validation techniques: RT-qPCR for mRNA expression levels, comparing with control markers (aggrecan and COL-2)

  • Imaging validation: X-ray, MRI, and H&E staining of NP tissue sections

  • Mechanistic studies: Examine MAPK8's relationship with autophagy pathways and immune cell infiltration

The study demonstrated that unlike other identified hub genes, MAPK8 expression was consistently elevated in IDD, suggesting its potential as a therapeutic target for this condition.

What is the emerging role of MAPK8 in lung cancer, and how can researchers effectively study this connection?

A 2024 Nature study revealed MAPK8's important role in lung adenocarcinoma (LUAD) progression:

These findings suggest that targeting the miR-147b/DUSP8/MAPK8 axis could provide novel approaches for lung cancer treatment.

How does MAPK8 regulate autophagy, and what are the contradictions in current research findings?

Research on MAPK8's role in autophagy presents some contradictory findings:

• Supporting evidence for MAPK8's essential role:

  • Initial studies reported that MAPK8/JNK1 (but not MAPK9/JNK2) is required for starvation-induced autophagy

  • Proposed mechanism: BCL2 phosphorylation (on Thr69, Ser70, and Ser87) by MAPK8 disrupts BCL2-BECN1 interaction

  • This initiates BECN1-dependent autophagy

  • Multiple studies have supported this role

• Contradicting evidence:

  • Some studies found MAPK8/JNK1 and MAPK9/JNK2 are not required for autophagy caused by starvation or MTOR inhibition in murine fibroblasts and epithelial cells

  • MAPK9/JNK2 may also play contributing roles in autophagy

  • In primary hepatocytes, MAPK8/9 appears to suppress rather than promote autophagic flux

  • Pharmacological inhibition with JNK-IN-8 increased autophagic flux in some contexts

• Reconciling the contradictions:

  • The role of MAPK8 in autophagy appears to be highly context-dependent

  • Cell type, experimental conditions, and stress stimuli may determine whether MAPK8 promotes or inhibits autophagy

  • MAPK8 may serve a redundant role in BCL2 phosphorylation in certain cellular contexts

Researchers investigating MAPK8's role in autophagy should carefully design experiments with appropriate controls and consider multiple cell types to address these contradictions.

What are the considerations for using MAPK8 antibodies in multiplexed immunoassays?

When developing multiplexed immunoassays for MAPK8:

• Antibody pair selection:

  • Ensure epitopes recognized by capture and detection antibodies do not overlap

  • Validate antibody pairs to confirm they don't interfere with each other's binding

  • Consider using monoclonal antibodies with defined epitopes for highest specificity

• Cross-reactivity mitigation:

  • Test for cross-reactivity between antibodies in the multiplex panel

  • Implement extensive blocking procedures to minimize non-specific binding

  • Use species-matched negative controls for each antibody

• Signal separation strategies:

  • For fluorescence-based detection: Choose fluorophores with minimal spectral overlap

  • For chromogenic detection: Ensure signal separation through distinct substrate reactions

  • Consider sequential detection protocols if cross-reactivity cannot be eliminated

• Validation approaches:

  • Compare multiplex results with single-plex assays using the same antibodies

  • Include appropriate positive controls (e.g., stimulated cell lysates)

  • Validate with samples of known MAPK8 expression/activation status

• Data normalization:

  • Include internal reference proteins for normalization

  • Account for potential signal crosstalk during data analysis

  • Consider using machine learning approaches for signal deconvolution in complex samples

What are common causes of non-specific binding when using MAPK8 antibodies, and how can they be addressed?

Non-specific binding is a common challenge with MAPK8 antibodies. Here are potential causes and solutions:

For phospho-specific MAPK8 antibodies, additional considerations include maintaining phosphatase inhibitors throughout sample preparation and optimizing stimulation conditions to ensure robust phosphorylation .

How can researchers distinguish between MAPK8/JNK1, MAPK9/JNK2, and MAPK10/JNK3 when using antibodies?

Distinguishing between JNK family members requires careful antibody selection and validation:

• Antibody selection strategies:

  • Use antibodies raised against unique peptide sequences specific to each JNK isoform

  • Select antibodies that have been specifically validated against all three JNK proteins

  • Consider using antibodies designed to recognize specific splice variants

• Validation approaches:

  • Test antibodies on samples with known expression patterns of specific JNK isoforms

  • Use genetic models (knockouts or knockdowns) for each JNK family member

  • Employ overexpression systems with tagged versions of each JNK for positive controls

• Technical considerations:

  • Western blot can distinguish JNK isoforms by molecular weight differences:

    • JNK1/MAPK8: 46 kDa (p46) and 54 kDa (p54)

    • JNK2/MAPK9: 55 kDa

    • JNK3/MAPK10: 46 kDa

  • For IHC/IF, validate specificity through co-staining with isoform-specific markers

  • Consider using RNA methods (RT-PCR, RNA-seq) as complementary approaches to confirm protein findings

• Common pitfalls:

  • Many commercial antibodies show cross-reactivity between JNK family members

  • Post-translational modifications can alter mobility in gels

  • Tissue-specific expression patterns may complicate interpretation (JNK3 is predominantly expressed in brain)

How can researchers apply MAPK8 antibodies in single-cell analysis of heterogeneous tissue samples?

Single-cell analysis of MAPK8 in heterogeneous tissues presents both challenges and opportunities:

• Single-cell immunostaining approaches:

  • Multiplex immunofluorescence with MAPK8 antibodies and cell-type markers

  • Mass cytometry (CyTOF) using metal-conjugated MAPK8 antibodies

  • Imaging mass cytometry for spatial context within tissues

  • Proximity ligation assays to detect MAPK8 interactions at single-cell level

• Single-cell sequencing integration:

  • Correlate protein measurements with transcriptomic data using CITE-seq

  • Apply computational methods to integrate antibody-based measurements with RNA profiles

  • Use pseudotime analysis to track MAPK8 activity changes during cellular processes

• Spatial considerations:

  • Implement multiplexed immunohistochemistry with MAPK8 antibodies

  • Apply digital spatial profiling technologies

  • Correlate MAPK8 status with tissue microenvironment features

• Challenges and solutions:

  • Signal sensitivity: Use signal amplification methods (tyramide signal amplification)

  • Antibody specificity: Validate thoroughly in simple systems before applying to complex tissues

  • Data integration: Develop computational frameworks to integrate protein and RNA data

Recent applications in intervertebral disc degeneration research have shown that MAPK8 expression correlates with specific immune cell infiltration patterns, demonstrating the value of examining this signaling molecule in a cell type-specific context .

What are emerging applications of MAPK8 antibodies in therapeutic development and monitoring?

MAPK8 antibodies are increasingly important in therapeutic development:

• Target validation:

  • Use MAPK8 antibodies to confirm target engagement in drug development

  • Apply for pharmacodynamic biomarker development

  • Validate functional consequences of MAPK8 pathway modulation

• Patient stratification:

  • Develop companion diagnostics using MAPK8 antibodies

  • Identify patient subgroups likely to respond to JNK pathway modulators

  • Monitor MAPK8 activation status as a predictive biomarker

• Therapeutic monitoring:

  • Track MAPK8 phosphorylation status during treatment

  • Develop multiplexed assays to monitor multiple nodes in the pathway

  • Use in clinical trials to establish pharmacokinetic/pharmacodynamic relationships

• Emerging therapeutic areas:

  • Intervertebral disc degeneration: MAPK8 as a potential therapeutic target

  • Lung adenocarcinoma: Targeting the miR-147b/DUSP8/MAPK8 axis

  • Autophagy modulation: Context-dependent intervention in MAPK8 signaling

• Technological advances:

  • Antibody-drug conjugates targeting MAPK8-expressing cells

  • Intrabodies directed against active conformations of MAPK8

  • Nanobody development for improved tissue penetration and target access

These emerging applications highlight the growing importance of high-quality, well-validated MAPK8 antibodies in both basic research and translational medicine contexts.

What quality control measures should researchers implement when using MAPK8 antibodies?

To ensure reliable results with MAPK8 antibodies, implement these quality control measures:

• Documentation and record-keeping:

  • Maintain detailed records of antibody source, catalog number, lot number, and validation data

  • Document all experimental conditions, including incubation times, temperatures, and buffer compositions

  • Create a laboratory antibody validation database

• Routine validation:

  • Test each new antibody lot against a reference standard

  • Include appropriate positive and negative controls in every experiment

  • Periodically reassess antibody performance, especially after prolonged storage

• Application-specific controls:

  • Western blot: Include molecular weight markers and positive control lysates

  • IHC/IF: Include known positive tissues and secondary-only controls

  • IP: Perform IgG control pulldowns alongside target pulldowns

• Storage and handling:

  • Aliquot antibodies to minimize freeze-thaw cycles

  • Store according to manufacturer recommendations (typically -20°C)

  • Monitor for signs of degradation or contamination

• Reporting standards:

  • Follow the minimum information about antibody experiments guidelines

  • Provide complete antibody information in publications

  • Share validation data via repositories or supplementary materials

Implementing these quality control measures will significantly enhance reproducibility and reliability of MAPK8 antibody-based experiments.

What methodological considerations should guide experimental design when studying MAPK8 in different cellular contexts?

When designing experiments to study MAPK8 across different cellular contexts:

• Cell type considerations:

  • Account for tissue-specific expression patterns of MAPK8 isoforms

  • Consider basal activation state of JNK pathway in different cell types

  • Adapt lysis conditions to cell type (adherent vs. suspension, primary vs. cell line)

• Stimulus selection:

  • Choose physiologically relevant stimuli for the cell type under study

  • Establish appropriate time courses (JNK activation can be transient)

  • Consider combinatorial stimuli to mimic complex in vivo conditions

• Detection strategy optimization:

  • Select antibodies validated in your specific cell type

  • Optimize fixation conditions for each cell type (especially for primary cells)

  • Consider subcellular localization analysis (nuclear translocation upon activation)

• Context-dependent interpretation:

  • MAPK8's role in autophagy varies by cell type and context

  • Different pathological conditions may show distinct MAPK8 activation patterns

  • Consider parallel analysis of upstream activators and downstream targets

• Translation between models:

  • Validate findings across multiple model systems (cell lines, primary cells, tissues)

  • Consider species differences when translating between animal models and human studies

  • Develop consistent protocols that work across experimental systems