MAPKAPK5 Antibody

What is MAPKAPK5 and what are its primary functions in cellular pathways?

MAPKAPK5, also known as PRAK, is a tumor suppressor serine/threonine-protein kinase involved in mTORC1 signaling and post-transcriptional regulation. It belongs to the CAMK Ser/Thr protein kinase family and contains conserved protein kinase domains I through XI .

As a major stress-activated kinase, MAPKAPK5:

• Phosphorylates multiple substrates including FOXO3, ERK3/MAPK6, ERK4/MAPK4, HSP27/HSPB1, p53/TP53, and RHEB

• Acts as a tumor suppressor by mediating Ras-induced senescence and phosphorylating p53/TP53

• Functions as a negative regulator of mTORC1 signaling by mediating phosphorylation and inhibition of RHEB

• Participates in post-transcriptional regulation of MYC via FOXO3 phosphorylation

MAPKAPK5 is activated through phosphorylation by MAP kinases including MAPK1/ERK, MAPK14/p38-alpha, and MAPK11/p38-beta in response to cellular stress and proinflammatory cytokines .

What experimental applications are validated for MAPKAPK5 antibodies?

MAPKAPK5 antibodies have been validated for multiple research applications as shown in the following table:

Published reports demonstrate successful application in knockdown/knockout validation studies as well .

How should researchers confirm the specificity of MAPKAPK5 antibodies?

Antibody specificity should be validated using at least one of these evidence-based approaches:

• Genetic Knockout/Knockdown Validation: Using CRISPR/Cas9 system to create MAPKAPK5 knockout cell lines or siRNA knockdown, researchers should observe absence of signal in KO/KD samples compared to wild-type controls .

• Orthogonal Validation: Compare antibody-based detection with antibody-independent detection methods (e.g., MS proteomics) across multiple samples to verify correlation of expression patterns .

• Recombinant Expression Validation: Test antibody against cells with overexpressed MAPKAPK5 versus control cells .

• Independent Antibody Validation: Use multiple antibodies targeting different epitopes of MAPKAPK5 to confirm consistent results .

• Capture Mass Spectrometry: Excise gel bands from Western blot at migration size and confirm presence of MAPKAPK5 peptides by MS analysis .

The Human Protein Atlas demonstrated successful validation of over 6,000 antibodies using these methods, establishing them as reliable standards for antibody validation .

What is the expected molecular weight of MAPKAPK5 in Western blot applications?

MAPKAPK5 should be detected at 54 kDa in Western blot applications. Both the calculated and observed molecular weight in validated studies is 54 kDa .

Researchers should note that MAPKAPK5 has two isoforms produced by alternative splicing , which might result in slightly different band patterns depending on the cell type and antibody epitope.

What cell lines are optimal for studying MAPKAPK5 expression and function?

Based on validated antibody testing and research applications, these cell lines are recommended:

For Cell-Based ELISA applications, the minimal detection threshold was established at approximately 5,000 HeLa cells per well, with optimal results when cells are 75-90% confluent (approximately 30,000 cells per well for overnight culture) .

What are critical factors affecting phosphorylation status of MAPKAPK5 that might impact antibody detection?

MAPKAPK5 undergoes several phosphorylation events that researchers should consider:

• Activation phosphorylation site: Thr-182 is the regulatory phosphorylation site located on the T-loop/loop 12. This site is phosphorylated by ERK3/MAPK6 or ERK4/MAPK4, leading to activation .

• Subcellular localization determinant: Ser-115 phosphorylation by PKA/PRKACA leads to localization to the cytoplasm .

• Autophosphorylation: MAPKAPK5 undergoes autophosphorylation which may affect epitope accessibility .

• Phosphorylation by p38 MAPK pathways: Phosphorylation at Thr-182 by p38-alpha/MAPK14 and p38-beta/MAPK11 is reported but subject to debate in literature .

These phosphorylation events can affect antibody binding, especially if the antibody epitope contains or is adjacent to a phosphorylation site. For studying specific phosphorylated forms, phospho-specific antibodies should be used.

How can researchers apply MAPKAPK5 antibodies to study protein-protein interactions?

For investigating MAPKAPK5 protein-protein interactions, multiple approaches have been validated:

• Proximity Ligation Assay (PLA): This technique has been successfully used to visualize and quantify interactions between MAPKAPK5 and EIF4EBP1. The protocol uses anti-MAPKAPK5 rabbit purified polyclonal antibody (1:1200 dilution) paired with antibodies against interaction partners .

• Co-Immunoprecipitation: IP-WB antibody pairs have been validated for studying MAPKAPK5 interactions. These specialized sets contain one antibody for immunoprecipitation and another to detect the precipitated protein complex in western blot .

• Cell-Based ELISA: This approach can be used to study how various treatments affect MAPKAPK5 interactions with other proteins within intact cells, providing insights into the dynamics of protein-protein interactions in response to stimuli .

When designing experiments to study MAPKAPK5 interactions, consider its known interaction partners including HSP27/HSPB1, ERK3/MAPK6, ERK4/MAPK4, and DJ-1 which is known to prevent oxidative stress-induced cell death .

What methodological approaches should be used when investigating MAPKAPK5's role in stress responses and tumor suppression?

To investigate MAPKAPK5's function in stress responses and tumor suppression, researchers can employ these validated methodological approaches:

• Cellular stress induction protocols:

  • Treatment with cellular stressors and proinflammatory cytokines to activate p38 MAPK pathways

  • UV exposure and serum starvation to trigger stress responses

  • Doxorubicin treatment to study MAPKAPK5 degradation and its negative regulation of doxorubicin-induced apoptosis in hepatocellular carcinoma cells

• Functional assays:

  • HSP27 phosphorylation status as a downstream readout of MAPKAPK5 activity

  • F-actin rearrangement following PKA-induced MAPKAPK5 activation

  • Monitoring RHEB phosphorylation to assess mTORC1 pathway inhibition

  • Analysis of FOXO3 nuclear localization and subsequent miR-34b/c expression to evaluate MYC translation inhibition

• Tumor suppressor function evaluation:

  • p53/TP53 phosphorylation and Ras-induced senescence assays

  • Cell proliferation and apoptosis measurements in normal versus stressed conditions

  • Evaluation of MAPKAPK5's role in hepatocellular carcinoma through extracellular vesicle-mediated transfer of lncRNA MAPKAPK5-AS1

Recent research has identified MAPKAPK5-AS1 as a contributing factor to hepatocellular carcinoma proliferation when delivered by carcinoma-associated fibroblasts-derived extracellular vesicles (CAF-EVs), offering a new angle for MAPKAPK5-related cancer research .

What are common technical issues when working with MAPKAPK5 antibodies and how can they be resolved?

For Cell-Based ELISA applications specifically, normalization methods are crucial for reliable results:

• Use anti-GAPDH antibody as internal positive control

• Apply Crystal Violet whole-cell staining to normalize for cell density

• For phosphorylated targets, normalize using antibodies against non-phosphorylated counterparts

How can researchers effectively use MAPKAPK5 antibodies in cancer research applications?

Recent studies have identified MAPKAPK5 as a significant player in cancer biology, particularly in hepatocellular carcinoma (HCC). Researchers can leverage MAPKAPK5 antibodies for cancer research through these approaches:

• Tumor suppressor function investigation:

  • Analyze MAPKAPK5 expression levels across cancer types using validated antibodies in IHC, WB, or ELISA

  • Compare expression between tumor and adjacent normal tissues

  • Correlate expression with clinical features and outcomes

• Mechanistic studies:

  • Investigate MAPKAPK5's interaction with p53/TP53 in response to DNA damage

  • Study MYC regulation through FOXO3 phosphorylation and subsequent miR-34b/c expression

  • Analyze mTORC1 signaling modulation via RHEB phosphorylation

• Novel cancer pathway exploration:

  • Study the relationship between MAPKAPK5 and MAPKAPK5-AS1 (long non-coding RNA) in HCC

  • Investigate how MAPKAPK5-AS1 carried by CAF-EVs promotes HCC cell proliferation

  • Examine the role of MAPKAPK5 in doxorubicin resistance in hepatocellular carcinoma cells

Researchers studying HCC should consider that MAPKAPK5 degradation has been observed in response to doxorubicin treatment, where it appears to negatively regulate doxorubicin-induced apoptosis .

What considerations are important when selecting and validating MAPKAPK5 antibodies for phosphorylation state-specific analysis?

When analyzing specific phosphorylation states of MAPKAPK5, researchers should consider:

• Epitope selection considerations:

  • Verify the antibody epitope in relation to known phosphorylation sites (particularly Thr-182 and Ser-115)

  • For the polyclonal antibody targeting epitope region 192-206 (PQVLEAQRRHQKEKS), ensure this region doesn't contain phosphorylation sites that might interfere with binding

  • Consider phospho-specific antibodies for studying activation status specifically

• Validation strategies for phospho-state detection:

  • Use phosphatase treatment as negative control

  • Include activating treatments (stress inducers, p38 MAPK activators) as positive controls

  • Apply multiple validation pillars as described by enhanced validation protocols

  • Confirm subcellular localization changes correspond with phosphorylation status (nuclear to cytoplasmic translocation upon activation)

• Technical considerations:

  • Include phosphatase inhibitors in all sample preparation steps

  • Consider cell fixation methods that preserve phosphorylation status

  • Account for MAPKAPK5's dynamic localization (translocates to cytoplasm following phosphorylation and activation)

Researchers should note that phosphorylation at Thr-182 is the key regulatory modification that activates MAPKAPK5's kinase activity, while Ser-115 phosphorylation by PKA/PRKACA influences subcellular localization .

How can researchers design robust experiments to compare results from different MAPKAPK5 antibody clones and validate experimental findings?

To ensure robust experimental design when comparing results from different MAPKAPK5 antibody clones:

• Multi-antibody validation approach:

  • Use at least two independent antibodies targeting different epitopes of MAPKAPK5

  • Compare results from polyclonal antibodies (such as rabbit polyclonal) with monoclonal antibodies

  • When possible, include antibodies that have been validated by multiple methods (KO, orthogonal, etc.)

• Standardized experimental conditions:

  • Maintain consistent cell types, densities, and passage numbers

  • Standardize lysis conditions and protein quantification methods

  • Use identical blocking reagents and incubation times across experiments

• Comprehensive controls:

  • Include positive controls (cell lines with known MAPKAPK5 expression like HeLa, Jurkat)

  • Implement negative controls (knockout/knockdown samples where available)

  • Use loading controls (GAPDH, β-actin) for normalization

  • Consider blocking peptides to verify specificity

• Cross-validation with orthogonal methods:

  • Correlate antibody-based results with RT-qPCR data (transcript levels)

  • Validate key findings with mass spectrometry-based proteomics

  • Confirm functional results with genetic approaches (overexpression, knockdown)

The enhanced validation strategy by the Human Protein Atlas demonstrated that combining multiple antibody validation approaches significantly increases confidence in experimental results, with 1,630 antibodies validated by at least two methods and 267 validated by three or more methods .