Shb protein expression in control and Shb-transfected PC3 cells (A). Different clones of PC3 cells transfected with Shb and one control-transfected clone were subjected to Western blot analysis for Shb protein as previously described . Equal protein loading was assessed by ERK-protein analysis of the blot. Effect of Shb-overexpression on growth of control and Shb-overexpressing PC3 cells during 3 days (B). On day 0, cells from the different clones were trypsinized and counted in a hemocytometer, after which 10000 cells were plated in each well. Cell numbers were determined by counting in a hemocytometer on day one and three. 2-ME-induced apoptosis in control and Shb-overexpressing PC3 cells (C). Percentages of cells undergoing apoptosis as based upon DAPI nuclear staining and M30 staining were determined in three control clones and five clones overexpressing Shb after treatment (or not) with 10 μM 2-ME for 12 h. The experiments were carried out in duplicates in 3 separate experiments performed on different occasions, in which 200–600 cells from each treatment condition were counted at 40× magnification in a Zeiss immunofluorescence microscope. The mean values ± SEM are presented.