Fig. 3. ERK activation is similar in Wt and Rb-null cells and is sensitive to inhibition by U0126. Following overnight serum deprivation cells were treated with U0126 at a final concentration of 20 µM or vehicle control for 60 minutes. The cells were then stimulated with 10% serum for the indicated times. (a,c) Endogenous ERK activity was analysed by in vitro kinase assay. Equal amounts of cell lysates were subjected to immunoprecipitation followed by kinase assay using myelin basic protein (MBP) as the substrate. (b,d) Total cell lysates were analysed by western blotting for active phospho-ERK.