Over-expression of ubiquitinatable Pttg1 is required to elicit changes in the cell cycle protein Ube1. Presented are quantitative fluorescent Western blots of HEK293 cells (n = 3 for all proteins). (a) Changes in four cell cycle proteins known to be modified by Wldsafter transfection with either a Wldsconstruct (black bars) or a pituitary tumor transforming gene 1 (Pttg1) over-expression construct (white bars). The first portion of the graph shows normalized Pttg1 levels accounting for differences in transfection efficiency. Note how Pttg1 induced the same level of increase in ubiquitin-activating enzyme E1 (Ube1) expression as Wldsbut had no effect on the three other proteins. (b) changes in Ube1 can only be induced by a ubiquitinatable form of Pttg1, because transfection with a non-ubiquitinatable form of Pttg1 (gray bars) could not elicit any changes in Ube1 expression (right portion of graph).