Extended Data Fig. 5: fEMSA of NANOG WT and W8A mutant against various NANOG DNA targets.
H6f-NANOG WT-eGFP displays tighter and more cooperative binding to fluorescently labeled (AF647) DNA targets (a, 40 bp GATA6 (1 nM); b, 257 bp pRSAT satellite DNA (1 nM); c, 404 bp Nanog promoter (1 nM)) than W8A mutant. Two independent measurements were performed for each DNA target. (Right panels), corresponding plots quantifying the fraction bound vs NANOG concentration from the fEMSA data shown in a-c, respectively. Fraction bound DNA was determined by subtracting band intensities relative to 100% free DNA (without NANOG, lanes 1 and 9). Data represent 2 independent experiments. d, (Left panel), NANOG CTD does not specifically interact with DNA. EMSA of NANOG CTD (0–20 µM) with 1 µM GATA6-DNA. (Right panel), CTD (3.4 µM) aggregation kinetics monitored by ThT fluorescence with 1 µM GATA6-DNA. Data are presented as mean values +/- SD; n=3 independent replicates.
