Supplemental Material

This article contains the following supporting material:

• Figure 1 - Plk1 binds to BubR1, but not Mps1, in mitosis. The endogenous Mps1 and BubR1 were immunoprecipitated from thymidine- or nocodazole-arrested HeLa cell lysates and blotted with α-Plk1 (top panel), α-Mps1 (middle panel), and α-BubR1 (bottom panel). The α-GST IP was included as a negative control. The input lysates were also blotted with α-Plk1.

• Figure 2 - Depletion of BubR1 by RNAi does not significantly perturb the kinetochore localization of Bub1 and Plk1. (A) The time line of experiments described in Figures 5-7 and Supplementary data Figures S2-S4. RNAi transfection occurs at 0 hr. Thy, thymidine; Noc, nocodazole. (B) HeLa cells that were either mock transfected or transfected with siRNAs against Bub1, Plk1, or BubR1 were harvested and blotted with the indicated antibodies. (C) HeLa cells that were either mock transfected or transfected with siRNA against BubR1 were stained with α-Bub1, CREST, α-BubR1, and DAPI. In the merge, Bub1-staining is shown in green, CREST in red, and DAPI in blue. The scale bar indicates 5 μm. (D) The cells described in (C) were stained with α-Plk1, CREST, α-BubR1, and DAPI. In the merge, Plk1-staining is shown in green, CREST in red, and DAPI in blue. The scale bar indicates 5 μm.

• Figure 3 - The kinetochore localization of BubR1 is diminished in the Bub1- and Plk1-RNAi cells. (A) HeLa cells that were either mock transfected or transfected with siRNA against Bub1 were stained with α-BubR1, CREST, monoclonal α-Bub1, and DAPI. In the merge, BubR1-staining is in green, CREST in red, and DAPI in blue. The scale bar indicates 5 μm. (B) HeLa cells that were either mock transfected or transfected with siRNA against Plk1 were stained with α-BubR1, CREST, monoclonal α-Bub1, and DAPI. In the merge, BubR1-staining is in green, CREST in red, and DAPI in blue. The scale bar indicates 5 μm. (C) Quantification of BubR1 immunofluorescence signals at the kinetochores in cells described in (A) and (B).

• Figure 4 - The kinetochore localization of INCENP and Bub1 is diminished in Aurora B-RNAi cells. (A) HeLa cells that were either mock transfected or transfected with siRNA against Aurora B were stained α-INCENP, CREST, and DAPI. In the merge, INCENP-staining is shown in green, CREST in red, and DAPI in blue. (B) HeLa cells that were either mock transfected or transfected with siRNA against Aurora B were stained α-Plk1, α-Aurora B, CREST, and DAPI. In the merge, Plk1-staining is shown in green, CREST in red, and DAPI in blue. (C) Quantification of INCENP immunofluorescence signals at the kinetochores in cells described in (A). (D) Quantification of Plk1 immunofluorescence signals at the kinetochores in cells described in (B).