Figure 5.
In vivo ubiquitination of active c-Src. (A) Cos-7 cells were transiently transfected with pCMV4 vector alone (lane 1) or pCMV4 expressing c-Src (lane 2), E378G (lane 3), Myc-tagged ubiquitin (lane 4) alone, or in the combinations indicated (lanes 5–8). For each transfection, 1 mg of lysate was immunoprecipitated with polyclonal antibody (PAb) N16 (anti-Src), separated by SDS/PAGE, transferred to PVDF membrane, and probed with mAb 9E10 (anti-myc). Cells used for lanes 6 and 8 were treated with 50 μM Z-L3VS for 14 hr before harvesting. * indicates a background band. (B) csk−/− cells were treated with either 50 μM Z-L3VS in DMSO or DMSO alone as a negative control. One milligram of lysate was immunoprecipitated with PAb N16 (anti-Src), separated by SDS/PAGE, transferred to PVDF membrane, and probed with mAb Ubi-1 (anti-ubiquitin).