Fig. 1. Chimeric molecules containing Grb2 or EGFR. (A) YFP-tagged Grb2. Mutations in each chimeric molecule are indicated. GGGG; four-glycine linker. (B) C-terminally tagged EGFR. (C) Immunoblot of Grb2-YFP. Lysates of COS-7 cells transfected with either wild-type Grb2 or mutant Grb2 fused to YFP were analyzed by western blotting (lane 1, YFP alone; lane 2, wild-type Grb2-YFP; lane 3, SH2m-YFP; lane 4, NSH3m-YFP; lane 5, CSH3m-YFP; lane 6, NCSH3m-YFP). The blot was probed with anti-GFP antibody and reprobed with anti-Grb2 antibody. (D) Western blot of EGFR-CFP. Lysates of COS-7 cells expressing either CFP alone or EGFR-CFP were analyzed by western blotting. The blot was probed with anti-GFP antibody (lanes 1,2) and reprobed with anti-EGFR antibody (lanes 3,4). Arrows point to EGFR-CFP (upper band) and endogenous EGFR (lower band). Molecular weight marker proteins (kDa) are indicated at the left. (e) Grb2-YFP associates with EGFR in an EGF-dependent manner. Wild-type Grb2-YFP was immunoprecipitated by anti-GFP antibody from the lysate of A431 cells expressing wild-type Grb2-YFP either before or after EGF stimulation. The blot was probed with anti-GFP antibody (bottom) or anti-EGFR antibody (top). (F) Grb2-YFP constitutively associates with SOS, Cbl and dynamin II. GFP immunoprecipitates from lysates of A431 cells transfected with either YFP alone (lane 1) or wild-type Grb2-YFP (lane 2) were immunoblotted with either anti-SOS, anti-Cbl, anti-dynamin II or anti-GFP antibody.