Mitotic NE SUMOylation events. (A) Epifluorescence images of WT cells analyzed by anti-SUMO immunofluorescence (SUMO). Nuclear position is determined by DAPI staining. Arrowheads highlight SUMO along the NE. SUMOylated septin ring position is indicated by an arrow. Images were rendered using the unsharp mask filter in ImageJ. Nuclear levels of fluorescence were quantified using line scan intensities of equatorial optical sections through nuclei (e.g., see red lines) of interphase (unbudded or small budded) and mitotic (anaphase/telophase) cells as described in Materials and methods. Plots show average fluorescence intensity (SUMO-IF and DAPI) at multiple points along a 1.75-µm line for n = 25 nuclei per cell cycle phase. Note that the perimeter of the DAPI signal lies adjacent to the NE and peaks of SUMO-IF intensity in mitotic cells. Error bars represent SD. Bar, 2 µm. (B) Cells were arrested in G1-phase using α-factor. Following α-factor removal, cultures were sampled every 10 min and analyzed by Western blotting using antibodies directed against the proteins indicated on the right. Note, Clb2 levels peak in metaphase. Gsp1 is a loading control. Blue arrowheads highlight four prominent SUMOylated species in the 40–55-kD range that arise in mitosis and decay as cells enter G1-phase. Molecular mass markers are shown in kD.