Figure 5

ET-1 stimulates c-Src-dependent transactivation of EGFR/PI3K/Akt leading to MAPKs and c-Jun phosphorylation. (A) For c-Src phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of BQ788 (1 μM), GPAnt2 (1 μM), GPAnt2A (1 μM), AG1478 (1 μM), or LY294002 (300 nM). (B) For EGFR phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of BQ788, GPAnt2, GPAnt2A, or PP1 (100 nM). (C) For Akt phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of BQ788, GPAnt2, GPAnt2A, PP1, AG1478, or LY294002. (D) For MAPKs phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of PP1, AG1478, or LY294002. For COX-2 expression, cells were incubated with ET-1 (10 nM) for 6 h in the absence or presence of U0126 (1 μM), SB202190 (300 nM), or SP600125 (300 nM). (E) For c-Jun phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of BQ788, GPAnt2, GPAnt2A, PP1, AG1478, LY294002, U0126 (1 μM), SB202190 (300 nM), or SP600125 (300 nM). The cell lysates were collected and analyzed by Western blotting using an anti-phospho-c-Src, anti-phospho-EGFR, anti-phospho-Akt, anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-c-Jun, or anti-GAPDH (as an internal control) antibody. The figure represents one of three similar experiments (n = 3).