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. 2000 Apr;11(4):1129–1142. doi: 10.1091/mbc.11.4.1129

Figure 5.

Figure 5

Activated Gαo stimulates B-Raf activity. (A) Expression of Raf proteins in CHO cells. Raf-1, A-Raf, and B-Raf were immunoprecipitated from lysates of nontransfected cells and detected by immunoblotting with specific antibodies as described in MATERIALS AND METHODS. The molecular masses (kilodaltons) of marker proteins are shown to the left, and the positions of Raf-1, A-Raf, and B-Raf migration are indicated by arrows. (B–D) CHO cells transfected in 10-cm dishes were serum starved for 18 h and assayed for endogenous Raf-1, A-Raf, and B-Raf activities as described in MATERIALS AND METHODS. (B) Effect of EGFR stimulation on Raf activity. Cells were transfected with 5 μg of EGFR and incubated for 5 min in the absence or presence of 100 ng/ml EGF. Data represent means ± SE of two to three independent experiments performed in triplicate. *, Significantly higher than basal (p < 0.01 by unpaired t test). In similar experiments carried out with nontransfected CHO cells, basal B-Raf activity (106,370 ± 7071 cpm) was not significantly increased by EGF (102,468 ± 5748 cpm) (n = 4). (C) Effect of activated Gαo on B-Raf activity. Cells transfected with 5 μg of empty vector, Gαo-WT or Gαo-Q205L and, where indicated, 5 μg of EGFR were incubated for 5 min in the absence or presence of 100 ng/ml EGF. Values are means ± SE of three independent experiments performed in triplicate. EGF induced a significant increase in B-Raf activity in all transfected cells (p < 0.01 by unpaired t test). *, Significantly higher than values from cells transfected with vector or Gαo-WT (p < 0.01 by unpaired t test). (D) Effects of N17-Ras expression, PMA pretreatment, and PKC or PI3K inhibitors on Gαo-mediated B-Raf activation. Cells were transfected with 4 μg of empty vector or Gαo-Q205L, and, where indicated, 8 μg of N17-Ras. PMA (1 μM), wortmannin (25 nM), GF 109203X (1 μM), and LY 294002 (20 μM) were used as described in the legend to Figure 2. Results are expressed as percentages of inhibition of the increase induced by Gαo-Q205L and represent the mean ± SE of two experiments in duplicate determinations. Expression of N17-Ras, PMA pretreatment, and the various inhibitors did not modify B-Raf activity in cells transfected with vector. Additional assays showed that both PMA pretreatment and GF 109203X completely abolished the increase in B-Raf activity induced by 5-min stimulation with 1 μM PMA (∼50%).