Fig. 2

Transactivation of IGF-1R by activated Src. (a) H226B and H226Br cells were transiently transfected with empty or pcDNA3.1-Src (Y527F) vectors. (b) A549, H460, and H522 cells were serum-starved and then stimulated with EGF (50 ng/ml). (c) H520 cells were transfected with empty or pBabe-Puro EGFR WT vectors, treated with dasatinib (Dasa; 0.5 μM) for 2 h, and then stimulated with EGF (50 ng/ml) for 2 min. (d) A549 cells were transfected with scrambled (siCon) or Src siRNA (siSrc) and stimulated with EGF (50 ng/ml) for 5 min. (e) H226B cells were transfected with empty or pIRES2-EGFP-integrin β3 vectors, treated with dasatinib (Dasa; 0.5 μM) for 2 h, and then attached to fibronectin (FN)-coated dishes for 30 min. (f, g) In vitro Src kinase assay was performed using Src, either from recombinant protein (rSrc) or from immunoprecipitates (IP) from A549 cells untransfected (f) or from H226B cells transfected with wild-type or kinase-dead mutant Src (Y416F) (g), and recombinant IGF-1R (GST-IGF-1R) as a substrate. (h) H520 cells were transfected with empty, wild-type, or mutant IGF-1R (Y1135F)-expressing vectors. (i) A549 cells were serum-starved and then stimulated with IGF (100 ng/ml) for 5 minutes. (j) H1299 cells stably transfected with control- or IGF-1R shRNAs were stimulated with 10 % FBS for 5 minutes. (k) In vitro IGF-1R kinase assay was performed using IGF-1R immunoprecipitates (IP) from A549 cells and recombinant GST-Src as a substrate. The expression levels of the indicated proteins were determined by Western blot analysis