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.