FIGURE 4.

BubR1 can be modified by SUMO-1. A, mitotic cell lysates prepared from taxol-treated cells were immunoprecipitated with the anti-BubR1 antibody or with the control IgG. Immunoprecipitates, along with asynchronized and mitotic cell lysates, were blotted for BubR1 and SUMO-1. B, mitotic cell lysates prepared from taxol-treated (or vehicle-treated) cells were immunoprecipitated with the anti-BubR1 antibody or with the control IgG. Immunoprecipitates, along with interphase and mitotic cell lysates, were blotted with the antibody to ubiquitin. C, HeLa cells were transfected with FLAG-UBC9 and His₆-SUMO-1 for 24 h followed by treatment with nocodazole and/or caffeine for an additional 18 h. Equal amounts of cell lysates of various treatments were incubated with Ni-NTA resin. After extensive washing, proteins specifically bound to the resin, along with lysate inputs, were blotted for BubR1. D, HeLa cells were co-transfected with HA-BubR1 (or vector), FLAG-UBC9 and His₆-SUMO-1 for 48 h. Equal amounts of cell lysates were incubated with Ni-NTA resin. Proteins specifically bound to the resin, along with lysate inputs, were blotted for BubR1 and the HA tag.