Fig. 5

Determination of upstream kinases involved in the proteasomal degradation of cyclin D1 and CDK4 by PF. a HCT116 cells were pretreated with PD98059 (ERK1/2 inhibitor, 20 μM), SB203580 (p38 inhibitor, 20 μM), SP600125 (JNK inhibitor, 20 μM) or LiCl (GSK3β inhibitor, 20 mM) for 2 h, and then co-treated with PF (25 μg/ml) for 3 h (cyclin D1) or 10 h (CDK4). b HCT116 cells were treated with PF (25 μg/ml) for the indicated times. c HCT116 cells were pretreated with PD98059 (ERK1/2 inhibitor, 20 μM) or LiCl (GSK3β inhibitor, 20 mM) for 2 h, and then co-treated with PF (25 μg/ml) for 1 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against cyclin D1, CDK4, p-ERK1/2, total-ERK1/2, p-GSK3β, total-GSK3β or p-cyclin D1 (Thr286). Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without PF treatment