Figure 6. Overexpression of EL and LMW-E differentially deregulates PLK1 and leads to its mislocalization.

A, pTRE-EL and B, pTRE-LMW-E cells were synchronized at the G1/S transition by aphidicolin block, induced with Dox for 24 h, released for the indicated times, and subjected to IP with anti-PLK1. Kinase activity was measured using α–casein as a substrate. Coomassie-stained gel of IgG (CB) was used as a loading control. Right panels, PLK1 kinase activity in A and B was measured by densitometry and normalized to the activity in the uninduced condition. +/- SD obtained from at least three independent experiments. C, pTRE-LMW-E cells were treated with Dox for 24 h, including a 16 h nocodazole treatment, and analyzed by western blotting for cyclin E, PLK1, and vinculin. Kinase activity was measured as in A and the bands corresponding to the α-casein were quantitated and depicted as bar graphs. D, pTRE-LMW-E cells were induced to express LMW-E by treatment with Dox for 24 h or depleted of Cdc25C by treating with a Cdc25C-specific siRNA for 72 h. Cells were costained for cyclin E (red), PLK1 (green), and DNA (blue) (top panels) and for PLK1 (green) or Cdc25C (red) (bottom panels).