Figure 5
Protein stability of exogenous wild-type TβRI-HA and the DM1 variant in HEK293 cells. Panel A: Translation efficiency. A plasmid (1 μg) encoding for wild-type (WT) TβRI or DM1 was transcribed and translated in vitro using a coupled reticulocyte lysate system. 35S-labeled proteins were resolved on a 10% SDS-polyacrylamide gel and visualized by autoradiography. The control plasmid (C) encoded for luciferase polypeptide (61 KDa). Panel B: Half-lives of wild-type TβRI and DM1. Cells were transfected with a plasmid encoding for wild-type TβRI (WT) or the DM1 variant. After 24 h transfection, the transfectants were treated with 50 μg/mL cycloheximide (CHX) for the designed time courses, as indicated at the top. WT (15 μg) and DM1 (150 μg) transfectant lysates were analyzed by Western blotting using an anti-HA antibody. Panel C & D: Protein stability of DM1 and DM2 associated with proteosome-related degradation. Transfectants were treated simultaneously with CHX and 5 μM, 20 μM MG132, or DMSO (vehicle control) for 30 min in Panel C. Transfectants treated with CHX and 20 μM MG132 or 10 μM lactacystin (Lata) for 60 min were shown in Panel D. Protein levels were determined by Western blotting of WT, DM1, and DM2 transfectant lysates using an anti-HA antibody. The same blot was reprobed with β-actin antibody as a control.