Fig. 5. Inactivation of the Rb protein is blocked by U0126 and LY294002 in Wt MEFs. After plating cells were placed in growth medium containing 0.5% FCS overnight, rendering the cells quiescent. Cells were then treated with U0126 at a final concentration of 20 µM or LY294002 at a final concentration of 20 µM or vehicle control for 60 minutes followed by stimulation with 10% (v/v) FCS. An equal amount of total cell lysate was then subjected to western blotting for Rb using a pan-antibody that detects both the hypo and hyper phosphorylated forms of the protein.