FIG. 7.

Time course of BK-induced tyrosine phosphorylation of the EGFR and EGFR-dependent activation of MAPK. (A) COS-7 cells expressing the BKR were serum starved for 12 h and then stimulated with BK (100 nM) for increasing times as indicated. Immunoprecipitates (IP) of the endogenous EGFR (anti-EGFR MAb; E. Merck AG, Darmstadt, Germany) were resolved by SDS-PAGE (7.5% gel) transferred to PVDF membranes, and Western blotted (WB) with either antiphosphotyrosine (α-pY) MAb 4G10 (top panel) or anti-EGFR antibody (polyclonal; Santa Cruz) (bottom panel). The results shown are representative of four separate experiments. (B) COS-7 cells were cotransfected with BKR and HA-MAPK, serum starved for 12 h, and stimulated with 100 nM BK in the absence or presence of AG1478 (10 nM, 10-min preincubation) for the times indicated. Shown is a representative Western blot with a polyclonal anti-phospho-MAPK antibody (Promega), representing two independent experiments.