Time course of HeT-A Gag protein expression and proteasome treatment.
(A) For the time course study, wild type, M1, M2, and M3 cells were seeded in T-25 flaks. At 80% confluence, cells were treated with 500 µM CuSO4, and equal numbers of cells were removed every 24 hrs for 5 days. Collected cells were lysed in SDS-PAGE buffer, subjected to immunoblotting, and the protein was detected by anti-FLAG antibody. Each membrane was stripped and probed for actin as a loading control. (B) For the proteasome treatment study, cells were seeded and induced as above. 72 hrs post CuSO4 inductions wild type, M2, and M3 cells were treated with 10 µM of proteasome inhibitor (MG132). Equal numbers of cells were harvested for various time points and analyzed by immunoblot.