Fig. 5

Oncogenic Ras reduces the enhanced ubiquitination of TβRII by SPSB1 and hence stabilizes TβRII. 293 T cells (a, b) were transfected with indicated FLAG-SPSB1/pEF-BOS control vector together with v-Ha-Ras/pcDNA3 control vector and HA-TβRII ± MYC-ubiquitin. Cells (a) were lysed 48 h post-transfection. Cell lysates were immunoprecipitated (IP) with anti-TβRII antibody conjugated with protein G beads. 36 h post-transfection, cells (b) were exposed to cycloheximide (20 μg/ml) for indicated periods and lysed. In all cases, both whole cell lysates and immunoprecipatates were examined for indicated proteins by immunoblotting (IB). Relative intensity of each TβRII band (b) was qualitatively measured using ImageJ as described in Fig. 4. In all case, results are representative of experiments repeated at least once