Mapk kinase phosphoprotein

Multiplex electrochemiluminescence immunoassay kits (MAPK Kinase Phosphoprotein and Akt Signaling Panel Assay Whole Cell Lysate Kits) were purchased from Meso Scale Discovery (Rockville, MD) and used according to manufacturer instructions -with the exception of incubating the samples at 4°C overnight -to measure phosphoprotein levels. Electrochemiluminescent assays are highly selective and quantitative, produce low background, have a broad dynamic range of detection, and require relatively small volume of sample compared to ELISAs and Western blots (41, 42) . The antibody precoated plates allowed for the simultaneous quantification of the following phosphoproteins: pERK1/2 (combined p42 and p44 isoforms), pJNK, and pp38 (MAPK kit) and pAkt, pGSK-3β, and pp70S6K (Akt kit). Samples were run in technical replicates and plates were read with a Sector Imager 2400 (Meso Scale Discovery) using the Discovery Workbench 4.0 (Meso Scale Discovery). Phosphoprotein values were averaged across technical replicates and normalized to total protein concentrations for statistical analyses (as in (39, (43) (44) (45) ). Inter-and intra-assay coefficients of variation (CV) were below 10%. Phosphoprotein signal data were excluded when the CV between duplicates exceeded this threshold.