Biochemical characterization of the interaction of SH3:RGT. (A) The side-chain of Arg760 in the RGT peptide is important for its activation activity. A standard c-Src activity assay was used to test whether mutation of Arg760 to Ala in the RGT peptide will affect its activation activity toward c-Src kinase. In the control experiment, no peptide was added into the reaction mixture containing c-Src kinase. The reading of OD⁴⁵⁰, an indicator for the phosphorylation of the c-Src substrate in the reaction mixture, was used to quantify the activity of the kinase. Values are mean ± SE; n = 3. *P < .05 (Student t test), statistically significant compared with control experiment. (B) The YAGT peptide shows no binding preference toward the SH3 domain. The wild-type SH3 protein or mutants were incubated with an excess amount of biotinylated YAGT or YRGT peptide. (C) Pull-down assays of SH3:YRGT. His-tagged SH3 domain was incubated with the biotinylated YRGT peptide in the presence of increasing concentrations of untagged wild-type SH3, SH3G116R, or SH3G127R, respectively. Each mixture was incubated with strep-tactin Sepharose solution. Strep-tactin resin was harvested by centrifugation and rinsed thoroughly. Proteins, which remain bound to the resin, were subjected to Western blot analysis. In the control experiment, no untagged SH3 protein was included in the pull-down assay.