Fig. 3: Identification of Nanog subregions interacting with Rad51.

a Co-IP assay identified the regions of Nanog interacting with Rad51. Plasmids expressing HA-tagged Rad51 and GFP-tagged Nanog fragments were co-transfected into 293 cells. Cell extracts were subjected to GFP glutathione-sepharose beads, followed by western blotting with antibodies against HA and GFP. The upper panel: schematic diagram of Nanog domains. FL full-length Nanog, N N terminus of Nanog, DB DNA-binding homeodomain, C C terminus of Nanog. b Purified GST-tagged Nanog fragments were conjugated to Glutathione-sepharose beads to capture His-tagged Rad51. c Co-IP assay identified the subregions of C interacting with Rad51. Plasmids expressing HA-tagged Rad51 and GFP-tagged C fragments were co-transfected into 293 cells. Cell extracts were subjected to GFP glutathione-sepharose beads, followed by western blotting with antibodies against HA and GFP. The upper panel: schematic diagram of the subregions in the C terminus. CD1 C terminal domain 1, WR tryptophan repeat domain, CD2 C terminal domain 2. d Purified GST-tagged Nanog-C fragments were conjugated to Glutathione-sepharose beads to capture His-tagged Rad51. e C and CD2 activate γH2AX foci. HeLa cells overexpressing GFP-tagged Nanog fragments were stained with the anti-γH2AX antibody (red) and DAPI (blue). f Western blotting assay examined the expression levels of Nanog fragments. g γH2AX foci in the cells from e were counted and analyzed. The nuclei containing more than 10 foci were considered to be positive. The data are based on three independent repeats, and presented as mean ± SEM. ***p < 0.001; **p < 0.01; *p < 0.05 (Student’s t-test).