Fig. 3.

Basal expression of Ski in prostate cell lines. (A) Total RNAs were isolated and semi-quantitative RT–PCR was performed to determine the mRNA levels of Ski in prostate cell lines. L-19 was used as an internal control. No RT samples derived from the same RNAs were also included. (B) Western blot analysis of Ski protein levels in prostate cell lines. Total cellular proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted using anti-Ski antibody. (C) Ski (green) expression in PZ-HVP7, DU145 and PC3 prostate carcinoma cells. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Ski was predominately localized in cytoplasm of the cells; the basal expression of Ski was high in prostate cancer cell lines but low in PZ-HPV7 cells. (D) Western blot analyses of Ski in PZ-HVP7 and PC3 cells treated with MG132 (25 µM). Anti-β-actin antibody was served as internal controls. Quantitative analysis of Ski in PZ-HPV7 and PC3 cells with or without treatment with MG132 (25 µM) (lower panel) were relative to untreated control (designated as one) after normalization to the signal obtained with β-actin. Each bar represents mean ± SEM (n = 3). *Significantly different (P < 0.05) compared with untreated controls. (E) Tissue sections were deparaffinized, rehydrated and blocked for endogenous peroxidase. Sections were blocked using normal goat serum in PBS and incubated with Ski antibody overnight. Sections were washed and incubated with Alexa Flour 488 anti-rabbit for 30min, washed again, and incubated with 4′,6-diamidino-2-phenylindole. Sections were then washed and counterstained with hematoxylin and mounted using xylene mounting medium. Immunofluorescence and H&E staining were analyzed by Zeiss microscope using 40× magnification. Representative images of the control and cancer tissues are shown.