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New pathway web pages
Whether your MAPK pathway research involves basic research tools, cell-based assays, or comprehensive screening services, Invitrogen has solutions for you. See our new MAPK pathway web page for more information.

New MAPK pathway web page Empower your research today using Invitrogen’s comprehensive portfolio of products and services to investigate the MAPK pathway—everything from high-quality reagents for basic research and assay development to validated biochemical and cell-based assays, and world-class profiling and screening services. See our portfolio of MAPK pathway–associated reagents at www.invitrogen.com/MAPK.

The MAPK pathway couples intracellular responses to the binding of growth factors to cell surface receptors and other extracellular stimuli.  There are three major classes of MAPKs. Extracellular signal-regulated kinases (ERK1/2), mediate growth factor receptor, (e.g. epidermal growth factor receptor (EGFR) and hepatocyte growth factor (HGF) receptor cMET) and G-protein coupled-receptor (GPCR) signaling through Ras-Raf-MAPK kinase pathway.  The other two classes of MAPKs, c-Jun N-terminal kinases (JNK1, JNK2 and JNK3) and p38 kinases (α, β, γ, δ), mediate stress-induced signaling events (e.g. IL-1 stimulation, TNF-α stimulation, and UV radiation) that lead to apoptosis.

The introduction of methodologies that allow simultaneous measurement of interrelated markers has revolutionized the drug discovery process. Monitoring multiple proteins in a single sample and experiment saves time, labor costs and sample volume.  Understanding the role of ERK, JNK and p38 MAPK in response to stimuli can provide insight into the design of future disease therapies. Luminex® multiplex technology allows for simultaneous detection of these three key protein kinases in a single sample.  In addition to these markers, Luminex assays are available for the determination of downstream cellular responses as well as for the detection of cytokines, chemokines and growth factors known to activate this pathway.

The analytical sensitivity of the ERK1/2 [pTpY185/187] assay is < 0.5 units/mL. This was determined by adding two standard deviations to the median fluorescence intensity obtained when the zero standard was assayed 32 times. This sensitivity corresponds to the amount of ERK1/2 [pTpY185/187] extractable from approximately 4000 PMA-treated Jurkat cells using Cell Extraction Buffer (Invitrogen Cat. FNN0011). The assay was found to be at least twice as sensitive as Western blotting.

The analytical sensitivity of the JNK1/2 [pTpY183/185] assay is <1.1 units/mL. This was determined by adding two standard deviations to the mean median fluorescence units obtained when the zero standard was assayed 30 times. This sensitivity corresponds to the amount of JNK1/2 [pTpY183/185] extractable from approximately 1 x 104 sorbitol-treated Jurkat cells using NP40 Cell Lysis Buffer and a Sample Treatment Buffer pre-incubation step. The assay was found to be at least twice as sensitive as Western blotting.

The analytical sensitivity of the p38 MAPK [pTpY180/182] assay is <3.6 units/mL. This was determined by adding two standard deviations to the mean median fluorescence units obtained when the zero standard was assayed 30 times. This sensitivity corresponds to the amount of total p38 MAPK extractable from approximately 1 x 104 anisomycin-treated Jurkat cells using NP40 Cell Lysis Buffer and a Sample Treatment Buffer pre-incubation step. The assay was found to be at least twice as sensitive as Western blotting.

• Learn more about Luminex® assays.

A wide range of stimuli are known to induce activation of the mitogen-activated protein kinase (MAPK) pathway.  Stimuli such as growth factors, cytokines and environmental stress are transduced from cell surface receptors such as GPCRs and growth factor receptors to multiple tiers of protein kinases leading to cell proliferation, growth, differentiation and apoptosis.  In short, the MAPK pathway plays an important role in regulating the overall health of a cell.  The definition of cell health for any given experiment determines the question that needs answering.  Do cells have the energy to proliferate?  Are the cells actively proliferating?  Which ones? 

The three assays described here each provide a different snapshot of cell health: Are the cells metabolically active? (alamarBlue®); Have the cells proliferated? (CyQUANT® NF); Which cells are actively proliferating? (Click-iT® EdU).  alamarBlue® is a redox indicator that yields a colorimetric change and a fluorescent signal in response to metabolic activity.  It is a proven safe and non-toxic dye used for quantitative analysis of cell viability and cell proliferation for cytokine bioassays and in vitro cytotoxicity studies.  The CyQUANT® NF Cell Proliferation Assay Kit provides a fast and sensitive method for counting cells in a population and measuring proliferation in microplate format. This assay can be completed in one hour, with no washes, cell lysis, long incubations or radioactivity required, and it is not dependent on physiological activities that may exhibit cell number–independent variability.  The Click-iT® EdU Cell Proliferation Assay is a novel alternative to BrdU assays for measuring de novo DNA synthesis.  This new technology eliminates the need to denature DNA, providing a superior alternative to the standard BrdU antibody-based method for measuring cell proliferation by flow cytometry or imaging.  Together, these assays provide simple, reliable and innovative solutions for answering your questions about cell health.

Cross section through the mid-intestine of a zebrafish larva. A 5-day-old zebrafish larva was exposed to a 16 hr pulse of 400 μM EdU and then fixed, embedded in paraffin, cut into 7 μm sections, and processed for detection using the Click-iT® EdU Alexa Fluor® 488 Imaging Kit (C10083). Slides were subsequently stained with Alexa Fluor® 568 soybean agglutinin (SBA) and TO-PRO®-3 dye (T3605). Proliferating nuclei appear white due to labeling with both Alexa Fluor® 488 azide (green) and TO-PRO®-3 dye (blue). Goblet cells in the intestinal bulb, labeled with Alexa Fluor® 568 SBA, appear red. This 20x z-section image was captured using a CoolSNAP™ camera (Princeton Instruments) mounted on a Nikon D-Eclipse C1 confocal microscope and processed (including pseudocoloring) using Photoshop® software (Adobe, Inc.). Image submitted by Sarah Cheesman, Institute of Molecular Biology, University of Oregon, USA.

• Learn more about assays for monitoring cell health.

New themes have emerged from recent advancements in the understanding of GPCR biology.  It is now clear that there is a great deal of complexity in downstream signaling events emanating from GPCR activation, including activation of mitogenic signaling (MAPK) pathways.  MAPK activation through GPCRs can be mediated by numerous mechanisms, including both secondary messenger-dependent and independent pathways (not involving Gα).  In this context, functionally selective compounds capable of inducing distinct signaling pathways may be valuable for drug discovery.  Therefore, measurement of intracellular MAPK activation provides a highly valuable functional endpoint for analysis of GPCR signaling. 

Until now, HTS-compatible technologies for measuring GPCR directed MAPK activation have been lacking.  To address this need, we have applied LanthaScreen™ Cellular Assay technology in combination with BacMam-mediated gene delivery to the development of a versatile platform technology for measuring ERK2 phosphorylation as a consequence of GPCR activation. We have validated this approach for the measurement of ERK2 activation across a variety of GPCRs, including Gαq, Gαi/o, and Gαs- coupled receptors.  In addition, this technology is compatible with a variety of cell backgrounds, without the need for modified receptors or chimeric G-proteins, providing a distinct advantage over other HTS-compatible technologies for measuring GPCR activation.  Together, these technologies provide a simple, versatile platform technology for GPCR functional selectivity studies and HTS screens.

Measurement of EGFR-mediated ERK2 Activation using the Lanthascreen™ ERK2 Cellular Assay.  U2OS cells were transduced using BacMam GFP-ERK2 virus.  On the following day, cells were transferred to a 384 well plate and serum starved.  On the third day, cells were stimulated with serial-diluted EGF for approximately 7 minutes.  Following stimulation, media were removed and cells were lysed with 20uL of LanthaScreen™ Cellular Assay Lysis Buffer in the presence of LanthaScreen™ Tb-anti-ERK2 [pThr 185 / pTyr 187] Antibody (PV5269).  Following equilibration at room temperature, the TR-FRET emission ratios were measured on a BMG Pherastar fluorescence plate reader.

Measurement of CCKAR Activation using the Lanthascreen™ ERK2 Cellular Assay.  The GeneBLAzer® CCKAR HEK 293T Cell Line (K1548) was transduced using BacMam GFP-ERK2 virus.  On the following day, cells were transferred to a 384 well plate and serum starved.  On the third day,cells were stimulated with serial-diluted Cholescytokin for approximately 7 minutes.  Following stimulation, media were removed and cells were lysed with 20uL of LanthaScreen™ Cellular Assay Lysis Buffer in the presenceof LanthaScreen™ Tb-anti-ERK2 [pThr 185 / pTyr 187] Antibody (PV5269).  Following equilibration at room temperature, the TR-FRET emission ratios were measured on a BMG Pherastar fluorescence plate reader.

• To learn how to gain early access to this new combined approach, contact us at discoverysciences@invitrogen.com.

The cellular remodeling that occurs during proliferation, growth, movement and invasion is possible due to microtubule reorganization.  Recent research has shown that members of the mitogen-activated protein kinase (MAPK) pathway, specifically MKK1 and ERK, regulate microtubule stability in a Ras dependent manner.  Along with its role played in microtubule stability, ERK has been shown to regulate the bidirectional transport of melanosomes for the distribution of pigment through cells.  At the very least, signaling through the MAPK pathway is tied to the regulation of microtubule structure and function.  The ability to visualize, in real time, the constant restructuring of microtubules may provide better understanding of the role played by different signaling pathways in this cellular process.

Our experience in developing targeted fluorescent reagents has allowed us to provide researchers with the most complete list of tools for monitoring microtubule dynamics.  We offer the Oregon Green® 514 conjugate of highly purified, bovine brain tubulin, the standard source of tubulin for research.  Molecular Probes exclusively provides three fluorescent derivatives of paclitaxel: Oregon Green® 488 paclitaxel, BODIPY® FL paclitaxel and BODIPY® 564/570 paclitaxel.  These fluorescent paclitaxel derivatives are promising tools for imaging microtubule formation and motility. Their fluorescent attributes should also make these conjugates useful reagents for screening compounds that affect microtubule assembly.  In addition to these fluorescent dye conjugates, we have new Cellular Lights™ reagents for the efficient transduction and robust expression of MAP4 and Tubulin fused to fluorescent proteins. These reagents enable visualization of microtubules in living or fixed cells and are ideal for studies of changes in cellular morphology, including cytoskeletal rearrangements or cell division.

U20S cells transduced with Organelle Lights™ Mito-OFP and Cellular Lights™ MAP4-GFP. U20S cells were transduced with Organelle Lights™ Mito-OFP (C36222) and Cellular Lights™ MAP4-GFP (C10105) and imaged the next day. Cells were imaged in McCoys media (Gibco Cat. 12330). Cells were imaged on a Deltavision core microscope with a 40X objective, images were acquired at 30 second intervals for 53 minutes. Fluorescence was detected using the appropriate filters (FITC/TRITC).

• Find out more about  our reagents for visualizing components of the cytoskeleton.