Fig. 2.

Kinase assay and Y419 phosphorylation of Src-V5 constructs. a Cells were transfected with Src-V5, Src(S75A)-V5, Src(S75E)-V5, and Src(Y530F))-V5, and cell extracts were immunoprecipitated with V5 antibody. Cell extract from non-transfected cells was similarly immunoprecipitated as a control. The kinase activity of the immunoprecipitated proteins was examined by in vitro kinase assay using a peptide substrate and the results were normalized to amount of V5-tagged protein present, as determined by immunoblotting (see panel b). Kinase activity of the non-transfected cells was insignificant (less than 10% of Src-V5), but is not plotted, as no V5 was detected. The relative kinase activity of the Src-V5 constructs is shown as a percentage of Src(Y530F)-V5. Statistical significance was evaluated using one-way ANOVA (*p < 0.05, n = 3). b Immunoprecipitated proteins assayed in a were immunoblotted to detect Src(pY419), Src(pY530), and V5. c The ratio of kinase activity to pY419 of Src-V5, Src(S75A)-V5, and Src(S75E)-V5 was plotted as a percentage of Src-V5. d A single flask of cells expressing Src-V5 was evenly divided into two dishes, which were incubated overnight in the presence or absence of olomoucine. Cell extracts were treated as in a. Kinase activity in the presence of olomoucine is plotted as a percentage of control. e Immunoprecipitated proteins assayed in d were immunoblotted to detect Src(pY419), Src(pY530), and V5. f The ratio of kinase activity to pY419 of Src-V5 in the presence of olomoucine was plotted as a percentage of Src-V5 in the absence of the inhibitor