1. Expression of dominant-negative RasN17 completely suppresses Ras activation in Rh1 cells.
Expression of dominant-negative RasN17 completely suppresses Ras activation in Rh1 cells. A, Rh1 or Rh1 cells infected with MSCV-I-GFP/RasN17 or control MSCV-I-GFP virus were serum-starved for 36 h, stimulated with IGF-I (10 ng/ml) for 5 min, and then lysed. Cleared lysates were incubated with glutathione-agarose beads bound to a GST-Raf1 RBD fusion protein (GST-RBD). The beads were then washed, and the proteins were resolved by SDS-PAGE. The amount of activated Ras bound to the GST-RBD beads was determined by anti-Ras immunoblotting (top panel). Cell lysates were also directly subjected to anti-Ras immunoblotting to determine levels of Ras in each sample (bottom panel). The blots are representative of experiments that were replicated three or four times. B, Rh1 cells or Rh1 cells infected with MSCV-I-GFP or MSCV-I-GFP/RasN17 were grown in MN2E medium for 24 h then incubated an additional 2 h with or without PD (15 μm). Subsequently they were stimulated with EGF. Phospho-Erk1 and phospho-Erk2 were detected after 5 min of stimulation with EGF (25 ng/ml; top panel) by Western blot using an anti-phospho-Erk1 and phospho-Erk2 antibody. The membranes were stripped and incubated with an antibody that recognized total Erk1 and Erk2 (bottom panel). The results that are shown are representative of those of two independent experiments. C, experimental details are as described for B, but cells were stimulated with IGF-I (10 ng/ml). The results of analysis with the anti-phospho-Erk1 and phospho-Erk2 antibody are shown in the top panel; the results of analysis with the anti-Erk1 and Erk2 antibody are in the center panel. Phosphorylation of Akt (Ser473) was detected after 5 min of stimulation with IGF-I. The membranes were stripped and incubated with anti-Akt antibody to ensure that equal amounts of protein were loaded in each lane. These results are representative of those of two independent experiments.
Kuntebommanahalli N. Thimmaiah et al. Cancer Res 2003;63:
©2003 by American Association for Cancer Research