In vitro kinase assay

In vitro kinase assays were conducted in the presence of 200 μM ATP, at 30 °C for 15 min. Briefly, GST-MEK1 or mutants were incubated in the absence or presence of active BRaf (V600E) Protein (Upstate). Changes in MEK1 phosphorylation were estimated by immunoblotting for p-MEK. To test the kinase activity of WT or mutant MEK1 protein, recombinant inactive ERK2 protein (GenWay Biotech) was used as a substrate and the reaction was terminated with the addition of 1XSDS loading buffer and boiling. Kinase activity was estimated by immunoblotting for pERK. When the subsequent in vitro kinase assay was performed after immunoprecipitation, 0.02% SDS was added to the wash buffer and one extra wash with kinase buffer was required.