Scs2 is required for, and is a target of, mitotic SUMOylation events. (A, C, and D) α-factor arrest-release assays were performed as in Fig. 1. Cell lysates from the indicated strains, at the times shown after release, were analyzed by Western blotting to detect SUMO conjugates, Clb2, and the Gsp1 load control (A, C, and D), as well as the V5₃ and HA₃ tags (D) as specified to the right of the blot. Note in panel D, for the ulp1^K352E/Y583H–V5₃ strains, the form of Scs2 produced in the cells examined is indicated above the lane. Red arrowheads point to SUMOylated Scs2 or SUMOylated tagged Scs2. Note that the WT SUMO conjugate profile shown in A is derived from that shown in Fig. 1 A. Blue arrowheads point to three other prominent mitotic SUMO conjugates. Molecular mass markers are shown in kD. (B) Epifluorescence images of WT, scs2Δ, and scs2^K180R (SUMOylation site mutant—SUMO site) cells analyzed by anti-SUMO immunofluorescence (SUMO) and DAPI staining are shown. Imaging and quantification were performed as outlined in the Fig. 1 legend. Mitotic cells are shown (note SUMOylated septin ring). The nuclear distribution of SUMO, in relation to DAPI-stained nuclear DNA, in mitotic cells from each strain was quantified using line scans (n = 25). Note, quantification of lines scans shown in B were obtained at the same time as data shown in Fig. 1 A, and the WT data are also shown here for comparison. Error bars represent SD. Bar, 2 µm.