(A) Exclusion of the SUMO-deficient mutant PTEN K254R from the nucleus. Flag-FITC immunofluorescence images of HEK293 cells transfected as indicated. Insets show DAPI staining. (B) Nuclear retention of SUMOylated PTEN. Immunofluorescence as in (A). Cells were treated with 10 ng/ml of leptomycin B for 4 hours. (C) Decreased nuclear PTEN localization following genotoxic stress. Immunofluorescence as in (A) of PTEN-FITC U87MG cells transfected as indicated, 4 hours post-IR. Bar graph represents percentage of cells with nuclear PTEN in control or IR-treated cells (p<0.001, t test, n=5, bars represent SEM). (D) Decreased nuclear PTEN following genotoxic stress. HeLa cells were treated as indicated, harvested after 4 hours and separated into cytoplasmic and nuclear fractions followed by immunoblotting for PTEN. Fractionation was monitored by immunoblotting for PARP (nuclear protein) and GAPDH (cytoplasmic protein). Arrow indicates SUMO-PTEN.