Figure 4

Src phosphorylates caspase-7 in vitro and interacts with caspase-7 in cells. (a) Src phosphorylates caspase-7 in vitro. Results of an in vitro kinase assay in the presence of [γ-³²P] ATP were visualized by autoradiography. Coomassie Brilliant Blue staining was used to assure that the same quantity of protein was loaded. Data shown are representative of results from triplicate independent experiments. (b) Src phosphorylates caspase-7 at tyrosine residues as determined by western blotting following an in vitro kinase assay. IB, immunoblot. (c) Src binds with caspase-7 but not caspase-3 in colon cancer cells. HT29 and SW480 total cell lysates (1 mg) were immunoprecipitated with anti-caspase-3/7, and the immunoprecipitated complex was probed to detect Src. (d) Src and caspase-7 co-localize in SW480 cells. SW480 cells were seeded in four-chamber glass slides and treated or not treated with 5-Fu (50 μM) for 48 h. Src and caspase-7 were detected by immunofluorescence staining with Src and caspase-7 primary antibodies, accompanied by the respective secondary antibody