Figure S1. Optimisation of an in vitro Bub1 kinase assay; Figure S2. Bub1 K795R does not support SAC function immortalised MEFs; Figure S3. Expression of GFP-H2B-Bub1C localises Bub1 activity to chromosome arms from Inhibitors of the Bub1 spindle assembly checkpoint kinase: synthesis of BAY-320 and comparison with 2OH-BNPP1
Creators: name: Amalina, Ilma
name: Bennett, Ailsa
name: Whalley, Helen
name: Perera, David
name: McGrail, Joanne C.
name: Tighe, Anthony
name: Procter, David J.
name: Taylor, Stephen S.
Publisher: name: The Royal Society
Year: 2021
Subjects: subject: Biochemistry
subject: Cell Biology
subject: 30306 Synthesis of Materials
subject: FOS: Chemical sciences
subject: 30503 Organic Chemical Synthesis

description: (A) Upper panel shows immunoblots for Bub1 and GFP using the input for immunoprecipitation, showing expression of the GFP-tagged wild-type and K821R Bub1 in HEK-293 cells when induced by overnight incubation with tetracycline. Tao1 was used as a loading control. In the lower panel, immunoblots for Bub1 and Bub3 show co-immunoprecipitation of GFP-Bub1 wild-type and K821R, with associated Bub3, using GST-tagged GFP binder protein. (B) In vitro kinase assay showing phosphorylation of H2B (H2Ap) and Bub1 autophosphorylation (Bub1-p) following incubation with immunoprecipitated wild-type, but not catalytically inactive K821R Bub1, in the presence of -32P-ATP. A Coomassie blue stain is included showing loading of H2A. (C) Optimisation of assay conditions by independent variation of parameters. From top to bottom panels show varying wild-type Bub1 enzyme concentration (volume of beads used from immunoprecipitation), H2B substrate mass, unlabeled ‘cold’ ATP concentration, Ci of -32P-ATP, and time (mins). Right hand panels show quantification of the signal from left panels, where values are normalised to one for the minimum detected H2A-p signal. IP, immunoprecipitation; Tet, tetracycline.; Line graph showing the proportion of cells in mitosis with monastrol treatment. Asynchronous BUB1F/ immortalised MEFs, expressing CRE recombinase, were pre-infected with indicated Bub1 adenoviruses and then treated with OHT for 24 hours. The time in mitosis is defined as the interval between nuclear envelope breakdown and chromosome de-condensation, as determined by phase-contrast time-lapse analysis. At least 16 cells were analysed for each condition. Images were taken every 2 mins for 24 hours. KD, deletion of Bub1 kinase domain; 38 deletion Bub3 binding domain of Bub1; DN, D919N; KR, K795R; OHT, 4-hydroxy-tamoxifen. Note that data shown as dashed lines are from ref. [14] and are for comparison only. ; (A) Immunoblot for GFP showing the expression of wild-type and D946N GFP-H2B-Bub1C in HeLa cells when induced by overnight incubation with tetracycline (+Tet). Tao1 was used as a loading control. (B) Immunofluorescence images of mitotic HeLa cells containing the tetracycline-inducible wild-type (left panels) or D946N GFP-H2B-Bub1C (right panels) transgenes, following treatment with nocodazole ± tetracycline for 16–18 hours and staining for DNA, H2ApT120 (pT120) and Sgo1. De-localisation of pT120 from centromeres to the chromosome arms is seen with expression of wild-type, but not D964N GFP-H2B-Bub1C (green highlighted panels). Scale bar, 5 m. (C) Immunofluorescence images of interphase HeLa cells containing the transgenes, with or without tetracycline treatment for 16–18 hours to induce fusion protein expression and staining for H2ApT120 (pT120). Cytoplasmic and nuclear staining of pT120 is seen in the presence of wild-type GFP-H2B-Bub1C expression and not in the presence of GFP-H2B-Bub1C D946N expression (green highlighted images). Scale bar, 10 m. Tet, tetracycline.