Down-regulation of cyclin D1 is the result of enhanced proteasomal degradation and is a GSK-3–dependent event. (A) Granta 519 and SP-53 cells were cultured in presence of the proteasome inhibitor LLnL 100 μM for 7 hours (top), and Granta 519 and Jeko-1 cells were grown for 24 hours in the presence of 25 μM LY294002, 1 μM LLnL, and their combination (bottom). Equal amounts of total-cell lysates were analyzed by immunoblotting for cyclin D1 protein. (B) Analysis of cyclin D1 protein expression in lysates from Granta 519 and SP-53 treated with 30 μM SB216763 (a GSK-3–specific inhibitor) alone and in combination with LY294002 (25 μM) or rapamycin (0.1 μM) for 30 hours. Tubulin shows equal loading of protein for each lane.