FIGURE 5.

BubR1 K250 is crucial for sumoylation. A, HeLa cells constitutively expressing His₆-SUMO-1 or His₆-SUMO-2, as well as parental HeLa cells, were collected and equal amounts of cell lysates were blotted with the antibody to the His₆ tag. These cells were also treated with taxol for 18 h after which cell lysates were prepared. Equal amounts of cell lysates were blotted for BubR1. B, schematic presentation of BubR1 (Wt) and its N-terminal fragment (610 amino acids) with (N-K250R) or without (N-Wt) K250 replaced with R250. GFP was fused in-frame with BubR1 N-terminal fragment. C, HeLa cells transfected with a plasmid construct expressing GFP-tagged N-terminal fragment of BubR1 (GFP-N-Wt) or its mutant counterpart (GFP-N-K250R) for 24 h followed by taxol treatment for 18 h. Equal amounts of cell lysates were blotted for BubR1, GFP, and β-actin. Arrow N-Wt-M denotes the sumoylated, GFP-tagged N-terminal fragment of BubR1. Arrow GFP-N denotes both the wild-type N-terminal fragment of BubR1 and its mutant counterpart. Vehicle and T+C denote lysate inputs that were derived from cells treated with vehicle or taxol (T) plus caffeine (C) for 18 h, respectively. D, HeLa cells constitutively expressing His6-SUMO-1 were transfected with His₆-HA-N-Wt or His₆-HA-N-K250R expression plasmids for 48 h. Ectopically expressed proteins were enriched by incubation with Ni-NTA resin and analyzed, along with lysate inputs, by Western blotting using the antibody to HA tag. His₆-HA-N-M denotes the sumoylated BubR1 N-terminal fragment.