Ubiquitination and Degradation of the Hominoid-Specific Oncoprotein TBC1D3 Is Mediated by CUL7 E3 Ligase
Figure 1
TBC1D3 is degraded and ubiquitinated.
(A) TBC1D3 degradation is inhibited by MG132. HeLa cells were transfected with Myc-TBC1D3. The cells were starved in serum-free DMEM with or without MG132 (20 µM) at 37°C for 3 h and then stimulated with 10% FCS. For the MG132 treatment group, MG132 was kept in the medium until the cells were collected. Cell lysates were resolved by SDS-PAGE and immunoblotted with anti-TBC1D3 and tubulin antibodies. TBC1D3 was substantially degraded over the 5 h incubation period, which was blocked by MG132 treatment. The number below each time point blot records the percent remaining normalized to the zero time. (B) TBC1D3 is ubiquitinated. Hela cells transfected with Myc-TBC1D3, were starved with DMEM containing 20 µM MG132 for 6 h and stimulated with 10% FCS for 20 min. The cell lysates were prepared under denaturing conditions and Myc-TBC1D3 was immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-ubiquitin antibody to visualize ubiquitinated TBC1D3 (top) or anti-TBC1D3 to visualize Myc-TBC1D3 (bottom). The migration of molecular weight standards is indicated. The experiments were repeated three times.
