WideScreen™ EpiTag™ ERK Data

WideScreen™ EpiTag™ Assays Overview Technology ERK Data Available Assays WideScreen™ RTK Assays RTK Assays RTK Data Available Assays WideScreen™ Biomarker Assays Human Cancer Panel 1 (Tumor Markers) Human Cancer Panel 2 (Growth Factors) Human CVD Panel 1 (Apolipoprotein) Human CVD Panel 2 (Inflammation) Human CVD Panel 3 Human CVD Panel 4 Human CVD Panel 5 (Acute Phase) Human CVD Panel 6

The first WideScreen™ EpiTag™ Assays have been developed to detect key signaling events in the ERK pathway from both human and mouse samples. ERK1 and ERK2 are serine/threonine kinases expressed broadly in tissues and various cell lines. ERKs are activated through their phosphorylation by the MAPK/ERK kinases MEK1 and MEK2. Once activated, ERK1 and ERK2 can phosphorylate many different proteins including cytoskeletal proteins, translation regulators, and transcription factors. ERK pathway protein levels and phosphorylation are deregulated in many types of cancer and are considered strong candidates for the development of therapeutic compounds.

 Standard Curve Multiplex

Three-fold serial dilutions of peptide and phosphopeptide standards were measured using the WideScreen™ EpiTag™ ERK Pathway Panel I Complete Kit. The EpiTag™ ERK Pathway Standards correspond to proteolytic fragments (1:1 ratio with target protein) generated during cell lysate digestion, and thus the standard curves can be used for molar quantitation of target proteins.

Specific Inhibition of ERK Phosphorylation after treatment with a MEK inhibitor

U87 cells were cultured in 96-well plates, serum-starved overnight, and treated with the MEK1/2 inhibitor SL327 at different concentrations for 1 h. Following rh-EGF stimulation (5 min), cell lysates were prepared, digested, and assayed using the listed EpiTag™ Bead Kits. Data shown are averages from triplicate culture wells (± SEM), expressed as % of uninhibited controls. Total protein levels are unaffected by inhibitor treatment, whereas ERK1 and ERK2 phosphorylation is inhibited with increased concentrations of inhibitor. The inhibitor dose response correlates closely with the expected SL327 IC₅₀ of ~200nM.

Stimulation and Inhibition of ERK Pathway Target Phosphorylation

Serum-starved A431 cells were left untreated, stimulated with rh-EGF (100 ng/ml, 5 min, Calbiochem Cat. No. 444939). EpiTag™ ERK Pathway Bead Kits were used to obtain quantitative measurements of the phosphoproteins shown (averages from triplicate culture wells ± SEM) MEK and ERK proteins are phosphorylated upon stimulation, whereas B-Raf phosphorylation at S446 is reported to be constitutive. The MEK 1/2 inhibitor SL327 strongly inhibits the phosphorylation of downstream ERK proteins.