Tyr326 and Tyr360 are the major phosphorylation sites of Src. A: CAP-EGFP and its mutant variants were transfected into HeLa cells together with c-Src and the phosphorylation of CAP was analyzed after immunoprecipitation. Tyr phosphorylation of the Y326F and Y360F mutant was reduced as compared to the WT CAP. An even lower level of phosphorylation could be detected for the Y326F+Y360F mutant. B: Quantification of the phosphorylation of CAP upon c-Src copexpression. Shown is the summary of the results of four independent experiments, error bars show standard deviation. C: c-Abl or c-Src was transfected into HeLa cells together with CAP-EGFP or EGFP, and the cells were treated with the Src inhibitor PP2, which does not inhibit Abl kinase at this concentration, or with non-inhibiting analogue PP3. In c-Abl transfected cells, inhibition of Src activity results in reduced phosphorylation of CAP, whereas in c-Src transfected cells, no phosphorylation could be detected after PP2 treatment.