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. Author manuscript; available in PMC: 2014 Mar 13.
Published in final edited form as: Oncogene. 2011 May 23;30(45):4590–4600. doi: 10.1038/onc.2011.166

Figure 4.

Figure 4

RasGRP3 is involved in Ras and Akt activation. (A) Individual cell lines were transiently transfected with RasGRP3 siRNA pool and Control siRNA pool 1 at 80 nM final concentration. After 72 hours, cells were treated with or without HGF (20 ng/mL) or EGF (100 pg/ml) as indicated for 20 minutes. Ras-GTP levels were detected by pull-down assay from 200 μg total protein. Levels of total Ras were used as control. Akt and phosphorylated Akt were detected by immunoblotting of cell lysates. (B) The Tet-on stable cell lines created from the M14 and SK-MEL-5 cell lines were treated with tetracycline (1 μg/ml) for120 hours. The cells were then treated with/without HGF (20 ng/mL) for 20 minutes. The Akt and phosphorylated Akt were detected by immunoblotting of cell lysates. (C) The Tet-on stable cell lines created from the M14 and SK-MEL-5 cell lines were treated with tetracycline (1 μg/ml) for 120 hours as indicated. Expression of c-Met was detected by immunoblotting of cell lysates. All results in this figure were representative of 3 independent experiments.