Regulation of Mcl-1 levels by proteasome inhibitors, etoposide, caspases and Bcl-2. (A) Jurkat cells were cultured in the presence or absence of epoxomicin (0.4 μM) or MG132 (0.5 μM) for the indicated amounts of time. Following preparation of cell lysates, cellular proteins were resolved by SDS-PAGE and Mcl-1 and γ-tubulin levels were determined by immunoblotting. (B) Following an 8-hour incubation with 0.4 μM epoxomicin, 25 μg/mL etoposide, or 100 ng/mL TRAIL, Jurkat cells were harvested and used for cell lysate preparation. Mcl-1 and γ-tubulin levels were determined by immunoblotting. (C) Jurkat cells were pretreated for 1 hour with 100 μM zVAD-fmk or regular medium and subsequently stimulated with 25 μg/mL etoposide or 0.4 μM epoxomicin. At the indicated times, cell lysates were prepared and Mcl-1 and γ-tubulin levels were determined by immunoblotting. (D) Bcl-2-overexpressing Jurkat cells and vector control cells were preincubated for 1 hour with or without 100 μM zVAD-fmk and subsequently stimulated with 0.5 μM MG132. Cells were harvested and cell lysates were prepared at the indicated times. Mcl-1, Bcl-2, and γ-tubulin levels were detected by immunoblotting.