Extended Data Fig. 2: NANOG purification and DNA binding activity.
a, Co-elution of Skp chaperone with h6g-NANOG WT by IMAC and SEC purification (Stain-free gel detection). NANOG W8A mutant did not co-elute with Skp, suggesting that the tryptophan residues mediate interactions between NANOG and Skp. b, IMAC purification of MBP-NANOG WT and W8A (Stain-free gel detection). Due to the presence of detergent, NANOG concentration was determined using BSA calibration. c, IMAC purification of h6f-NANOG WT/W8A-eGFP. NANOG concentration was determined from h6g-eGFP calibration. Green (GFP filter), Blue (Stain-free filter for all protein detection). d, e, Fluorescent EMSAs (fEMSA) with 5 nM GATA6-AF647 (green) and various concentrations of h6g-NANOG WT/Skp complex and MBP-NANOG WT. f, Fluorescently labeled purified SOX2, NANOG WT and W8A constructs. MW markers loaded in gels a-c,f are identical and comprised of 10,15, 20, 25, 37, 50, 75, 100,150, and 250 kD standards (as labeled in a). Data shown represent 2 independent experiments.
