Figure 3.

The evolutionary conserved cassette ABBA1-KEN2-ABBA2 in BUBR1 is essential for SAC signalling. (a) Alignment of ABBA1-KEN2-ABBA2 cassette (red). Linkers (black) between ABBA motifs and KEN2 are indicated by {n}. The sequence logo on top is representative for all eukaryotic sequences (colours reflect distinct amino acid properties and height of the letters indicates conservation of amino acids). (b) Schematic representation of LAP-hBUBR1 mutants. Colour coding is consistent for each mutant in this figure. (c) Immunoblots of BUBR1 and tubulin of mitotic lysates of HeLa-FlpIn cell lines stably expressing LAP-tagged BUBR1 proteins. Cells were treated with siRNA (40 nM) for 48 h and cells were released and arrested into Taxol after double thymidine block. (e) Time-lapse analysis of HeLa-FlpIn cells expressing hBUBR1 mutants, treated with 20 μM STLC. Data (N = 3 with n = 50 per experiment) indicate the mean of cumulative fraction of cells that exit mitosis after nuclear envelope breakdown. Transparent regions represent the standard error of the mean. Values between braces {} indicate the median value. Cells were scored by cell morphology using DIC imaging; see (d) for examples of SAC deficient (ΔABBA1/2) and proficient cells (wild-type). Only YFP-positive cells were considered for analyses. (f) Immunoblots of GFP, APC3 and CDC20 in LAP-BUBR1 precipitations (LAP-pulldown) in whole cell lysates of mitotic HeLa-FlpIn cells expressing LAP-BUBR1 mutant constructs. The mean and standard deviation values of three independent APC3/GFP co-immunoprecipitation experiments for all mutant LAP-BUBR1 cell lines are normalized to wild-type LAP-BUBR1 and depicted below the immunoblots.