Fig. 5. Effects of dominant negative Ras(Asn-17) expression on p21 by growth factors. Conditionally Ras(Asn-17) expressing NIH 3T3 cells were treated with 10 mM IPTG for 30–48 h to induce Ras(Asn-17) expression, and the cells were transferred to low serum. After a 4-h starvation, FGF-2 (5 ng/ml) or PDGF (10 ng/ml) were added for 2 h or TGF-ß1 (250 pM) for 12 h, followed by lysis, protein separation in 12.5% SDS-PAGE and immunoblotting with anti-p21, anti-phospho-ERK1/2, anti-ERK2, and anti-Ras antibodies as indicated. Cells cultured in low serum for 6 h in the absence of IPTG were used as a control to determine the fold induction of p21.