HEK293T cells were cultured in DMEM (Thermo Fisher: #12100046) supplemented with 10% FBS (Gibco: #10270106) at 37°C Temp and 5% CO2. Cells were transfected with pLVTHM-3Xflag-Oct4 plasmids (a kind gift from the Hans R. Schöler group) (40 (link)) using a 1:2 plasmid to polyethyleneimine (w/w) ratio (Polysciences: #23966). Fresh medium was changed 16 h after transfection. 72 h post transfection, cells were dissociated with 0.05% trypsin–EDTA and washed two times with DPBS and collected by centrifugation. Cell pellets were re-suspended in lysis buffer (20 mM HEPES–KOH pH 7.8, 150 mM NaCl, 0.2 mM EDTA pH 8.0, 25% glycerol, 1 mM DTT with cOmplete™ protease inhibitor cocktail (Roche: #11836145001) added fresh). Four freeze-thaw cycles in liquid nitrogen were used to lyse the cells. Lysates were centrifuged at 14 000 × g for 10 min at 4 °C and supernatants were collected and frozen. Large (18.5 × 20 cm) 6% native PAGE gels were used for EMSAs. Protein samples and fluorescently labeled DNA were incubated for 1 h at RT. For super shift EMSA, reactions were incubated with Oct4 antibody (Santa Cruz Biotechnology: #sc-8628) for an additional 30 minutes. Gels were pre-ran at 300 V for at least 1.5 h and for another 2.5 h to separate protein/DNA complexes.
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