Supplementary Figure 4: Identification of a Wapl-binding site on SA2–Scc1.
From: Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion
(a) Anti-Wapl and anti-tubulin blots of lysates of HeLa cells transfected with the indicated siRNAs with or without increasing amounts of the indicated GFP-Wapl plasmids. WT, wild type. The positions of the endogenous and GFP-Wapl are labeled. (b) Quantification of mitotic indices (defined as MPM2-positive, 4N cells) of cells in a. Error bars, range (n = 2, independent experiments). (c) Autoradiograph (top) and Coomassie stained gel (bottom) of 35S-labeled Myc-SA2–Scc1 proteins (input) and the same proteins bound to beads containing GST or increasing amounts of GST-Wapl-M. WT, wild type. (d) Quantification of the in vitro binding assays in c. The binding intensities were normalized to the amount of each input. Error bars, range (n = 2, independent experiments). The K290E and D326K mutants were only tested once.
