Fig. 2. Kinetics and MEK1(/2) dependency of p21 induction. NIH 3T3 cells were pretreated in low serum with or without PD 098059 (80 µM) for 4 h, after which FGF-2 (5 ng/ml; A), PDGF (10 ng/ml; B), or TGF-ß1 (250 pM; C) were added for the indicated times. Cell lysates were prepared followed by protein separation in 12.5% SDS-PAGE and immunoblotting with anti-p21, anti-p27Kip1, anti-phospho-ERK1/2, and anti-ERK2 antibodies as indicated. p21 levels were normalized against p27Kip1 levels and the fold induction as compared with nontreated cells starved for 4 h are shown below.