Cohesin-loader−stimulated cohesin loading.

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Cohesin-loader−stimulated cohesin loading. Cohesin-loader−stimulated cohesin loading. (A) Schematic of the cohesin loading assay. Circular DNA and cohesin, with or without the cohesin loader, are incubated in the presence of ATP. Cohesin-DNA complex are retrieved by immunoprecipitation using an antibody against the Pk epitope tag on the Smc1 subunit. The recovered DNA is analyzed by agarose gel electrophoresis. (B) Gel image and quantification of a cohesin loading time course experiment in the presence or absence of the cohesin loader. (C) The Scc2–Scc4 complex was analyzed by SDS–PAGE and Coomassie Blue staining next to the Scc2C fragment. (D) Gel image and quantification of recovered DNA from the cohesin loading assay performed with the indicated concentration of Scc2C in comparison with the Scc2/Scc4 complex. Mean values and standard deviations from three independent experiments are shown. Masashi Minamino et al. LSA 2018;1:e201800143 © 2018 Minamino et al.