Fig. 4.

Src attenuates ubiquitination by β-TrCP. (A) Autoubiquitination of TrCP is attenuated by Src. Ubiquitination of β-TrCP was determined using cotransfection of HEK293 cells with tagged-Ubiquitin (HA-Ub), active Src, and β-TrCP. Cells cotransfected with β-TrCP and Src show attenuated ubiquitination pattern. Cells were treated with 25 μM MG132 for 2 h to prevent proteasomal degradation of ubiquitinated substrates. (B) Taz ubiquitination by β-TrCP is attenuated in the presence of Src. HEK293 cells were cotransfected with Flag-R48K-Ub mutant, Flag-Taz, β-TrCP, and active Src (Y527F). Taz was immunoprecipitated, and its ubiquitination was observed using anti-ubiquitin antibody (P4D1). Cells were treated with 25 μM MG132 for 2 h. (C) Endogenous Taz ubiquitination is decreased by ouabain treatment. HEK293 cells were treated with 30 nM ouabain for 24 h. The next day, cells were treated with 25 μM MG132 for 2 h. Taz was immunoprecipitated, and its ubiquitination pattern was analyzed by Western blotting, as indicated.