Phosphorylated MDM2 resides mainly in the nucleus. SUDHL-1 cells were treated with either DMSO control or LY294002 (20 μM) for 24 hours, after which cells were prepared as cytospins and stained with fluorescently labeled antibodies against p(ser166)MDM2 and DAPI (A) or MDM2 and DAPI (B). SUDHL-1 cytospins were also analyzed for p53 expression and localization 24 hours after inhibitor treatment (C). Arrows point to cells in which p53 appears to be sequestered to cytoskeletal structures. MDM2:p53 complex formation in response to 24 hours treatment with 20 μM LY294002; vertical lines have been inserted to indicate a repositioned gel lane (D). Numbers represent the ratio of p53:MDM2 protein in arbitrary units after densitometric analysis. Equal quantities of cell lysate were entered into the IPs.