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. 2006 Feb 2;25(5):1093–1103. doi: 10.1038/sj.emboj.7600987

Figure 8.

Figure 8

The suppression of SRF and Egr-1 expression by oncogenic H-Ras is reversible. (A) NIH3T3tet-on/H-RasG12R cells were cultured in the presence of doxycycline for 48 h, after which the doxycycline was removed by washing. At the indicated time points, total cell lysates were prepared and Western blot analysis was performed with antibodies directed against H-Ras, phospho-Erk1/2 (Thr 202/Tyr 204), and cyclin D1. (B) NIH3T3tet-on/H-RasG12R cells were serum starved for 24 h in medium that contained 0.5% FBS, and then stimulated with PDGF for 1 h. Doxycycline was added before, 6 h after, or 48 h after PDGF stimulation (+Doxy). Doxycycline was removed by washing out the medium after culturing the cells with doxycycline for 48 h. The cells were cultured in the absence of doxycycline for 48 or 60 h, and then stimulated with PDGF for 1 h (Doxy-withdrawal). Serum starvation was performed before 24 h of PDGF stimulation. Total protein extracts were prepared and used to detect the expression of H-Ras, SRF, Egr-1, PTEN, and c-Fos by Western blot analysis. GAPDH was used as a loading control.