Fig. 2

Functional assays for BUB1 c.1965-1G>A and c.2296G>A (p.E766K) and 3D structure of the proteins and location of the identified BUB1 and BUB3 germline mutations. a Quantitative analysis of levels of BUB1 mRNA demonstrating reduced BUB1 expression in the c.1965-1G>A cell line compared to controls. Data are normalized against HPRT expression. Error bars represent standard error mean (SEM) values. b Quantification of chromosome segregation errors in EBV-transformed cells from a control individual and the BUB1 c.1965-1G>A mutation carrier. Measurements were performed in triplicate and error bars represent SEM values. c Localization and d quantification of BUB1 levels at the kinetochores of nocodazole-arrested EBV-transformed cells. Each dot represents one cell and the level of BUB1 is normalized to the kinetochore (KT) intensity of CENP-C and is the average fold change of three experiments (±SEM) normalized to the values of control cells. Cells from the c.1965-1G>A carrier revealed reduced levels of BUB1 at the KT in comparison to the control. ** P < 0.001; *** P < 0.0001. e Crystallographic 3D structure of BUB1 (a.a. 736–1083) and location of p.E766K and p.P825S, and 3D model of BUB3 (a.a. 6–324) and location of p.T26I (current study) and p.K21N, p.R149Q and p.F264L [2]. f Protein domains of BUB1 (UniProtKB - O43683) and BUB3 (UniProtKB - O43684) and location of the identified mutations. In red, residues affected by mutations identified in this study; in blue, residues affected by the mutations identified by de Voer et al. [2]; in orange, variants identified by Broderick et al. [4]; and in green, variants detected by Shindo et al. [6]