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. 2020 Sep 7;28(2):685–699. doi: 10.1038/s41418-020-00613-x

Fig. 1. Identification of Kap1 as an Oct4-binding protein.

Fig. 1

a Co-immunoprecipitation of Kap1 and Oct4 in mESCs. D3 mESCs extracts were incubated with control IgG, anti-Oct4, or anti-Kap1 antibodies, and the protein levels of Oct4 and Kap1 in the immunocomplexes were probed by western blot analysis. b Direct interaction of Kap1 and Oct4 in vitro. GST and GST-Kap1 proteins were incubated with His-Oct4 protein, followed by a pull-down with GSH agarose. The protein levels of GST and GST-Kap1 were confirmed by SDS-PAGE and Ponceau S staining. The His-Oct4 protein levels in the immunocomplexes and input were determined by western blot analysis. c Mapping the Kap1-binding domain in Oct4 protein. Schematic diagrams of full-length (FL) and deletion forms of Oct4 protein (upper panels). The Flag-tagged Oct4 constructs were overexpressed in 293FT cells and immunoprecipitated by anti-Flag antibody. d Mapping of Oct4-binding domain in Kap1 protein. Schematic representation of the HA-Kap1 FL and deletion mutants (upper panel). Flag-Oct4 was co-transfected with HA-Kap1 FL or its deletion mutants into 293FT cells, followed by immunoprecipitation with the anti-Flag antibody. The protein levels of Flag-Oct4 and Kap1 in immunocomplexes and lysates (input) were analyzed by western blotting. Arrows indicate the Kap1 FL and deletion mutants.