Fig. 7.

Effect of G9R mutation on p27 removal. (A) PC‐3 cells were transfected with pcDNA3.0 empty vector (CTRL), and pcDNA3.0 encoding p27 [WT], or G9R‐p27 [G9R], S10A/G9R‐p27 [S10A/G9R], and S12A/G9R‐p27 [S12A/G9R]. After 18‐h transfection, medium was changed and cells were treated with or without CHX (36 µm) for 6 h. After harvesting, cells were lysed and analyzed by western blotting using anti‐p27 mAb. The intensity of the specific signals was evaluated using imagej software, and value of the relative intensity over the CTRL (+CHX/−CHX) +CHX/CTRL is reported for each protein under analysis. (B) K562 cells were transfected with pcDNA3.0 encoding p27 (WT), G9R‐p27 (G9R), or S12D‐p27 (S12D). After 18‐h transfection, medium was changed and cells were treated with CHX (36 µm) for 0, 3, or 6 h. After harvesting, cells were lysed and analyzed by western blotting using anti‐p27 mAb. Actin was used as loading control. (C) K562 cells were transfected with pcDNA3.0 encoding p27 (WT) and G9R‐p27 (G9R). After 18‐h transfection, medium was changed and cells were treated as reported in the panel. After harvesting, cells were lysed and analyzed by western blotting using anti‐p27 mAb. Actin was used as loading control. (D) PC‐3 cells were transfected with pcDNA3.0 encoding p27 [WT], or G9R‐p27 [G9R], T187A/G9R‐p27 [T187A/G9R]. After 18‐h transfection, medium was changed and cells were treated with or without CHX (36 µm) for 6 h. After harvesting, cells were lysed and analyzed by 2D‐WB using anti‐p27 mAb. The intensity of the specific signals was evaluated using TotalLab CLIQS gel image analysis Software and the relative intensities of the monophosphorylated isoform [1P] over the unmodified isoform for each analysis are reported in the histograms at the bottom of the panel.