Figure 5
GUTK enhanced c-MYC protein degradation via increasing FBXW7 protein stability. Quiescent LNCaP (a and c) and PC-3 (b and d) cells were induced to re-enter the cell cycle in the absence or presence of GUTK at GI75 for indicated times. The total and phospho-c-MYC, total and phospho-GSK3β, total and phospho-ERK1/2, and FBXW7 α (110 kDa) and β (70 kDa) isoforms were analyzed by immunoblotting. The steady-state levels of FBXW7 mRNA in LNCaP (e) and PC-3 (f) cells during cell cycle re-entry with or without GUTK at GI75 were determined by RT-qPCR. The results are expressed as the mean±S.D. of triplicate assays. Quiescent LNCaP (g) and PC-3 (h) cells were induced to re-enter the cell cycle in the presence of 50 μM cycloheximide (CHX) with or without GUTK at GI75 for indicated times and the cell lysates were analyzed by immunoblotting. The plots were based on the mean value of three independent experiments, and the half-life (t1/2) of FBXW7 α and β isoforms was calculated by densitometric analysis using ImageJ software. α-Tubulin or GAPDH served as a loading control
