Fig. 4. Y4U32^527F is associated with the Triton X-100 insoluble cytoskeletal fraction. Mock-transfected CEF or cells expressing c-Src, Src^527F or Y4U32^527F were grown to confluence in 100 mm dishes. Cells were incubated in 1 ml of CSK buffer for 1, 4 or 10 minutes. The Triton-soluble fractions were collected, and the Triton-insoluble material at each time-point was solublized in RIPA buffer. 50 µg of cell lysates from each fraction were separated by 8% SDS-PAGE, transferred to PVDF membrane, blocked with 5% nonfat milk/TBS-T, and probed with rabbit anti-Src. Results are shown for mock-transfected CEF, c-Src, Src^527F and Y4U32^527F. C, CSK buffer; R, RIPA buffer. Cytoskeletal-associated proteins are defined in the R fraction.