(A) 2 days after siRNA transfection, cell lysates were analyzed by immunoblotting.
(B) PPC1 cells stably containing inducible shRNAs (#1, #2) were cultured for 2 days with (ON) or without (OFF) doxycycline. Protein lysates were analyzed by SDS-PAGE/immunoblotting.
(C) p27 expression levels in resected xenograft tumors. Tumor lysates were harvested after doxycycline treatment for 12 days. Relative p27 levels were quantified by densitometry analysis. ** p< 0.01
(D) PPC1 cells stably containing inducible shRNA (#2) were cultured with (Tet-ON) or without (Tet-OFF) doxycycline. After 48 hours, cells were treated with 25 μg/ml cyclohexamide (CHX), and cell lysates were harvested at the indicated times. Relative p27 expression was quantified by densitometry analysis, and the results expressed as the percentage of p27 expression at 0 hour. *** p < 0.001
(E) 48 hours after siRNA transfection, cells were fractionated to yield cytosolic and nuclear compartments, which were analyzed by SDS-PAGE/immunoblotting.