Mc480

For immunoprecipitation, cells were lysed on ice in TNE buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% Tergitol-type NP-40, 1 mM EDTA, and protease inhibitors). Anti-FLAG M2 magnetic beads (M8823, Sigma) were incubated overnight with cell lysate fractions. Samples were then washed 6 times with TBS. Western blotting was performed following standard principles and immunofluorescence was assessed using a Leica TCS SP2 spectral confocal microscope. Nuclear-cytoplasmic fractionation was performed following standard protocol. The following primary antibodies were used: anti-FLAG (F7425, Sigma), anti-NCoR (ABE251, Millipore), anti-HA (H6908, Sigma), anti-SMRT (ab24551, Abcam), anti-HDAC7 (ab12174, Abcam), anti-E-cadherin (BD Biosciences), anti-SSEA-1 (MC480, Cell Signaling), anti-SOX2 (MAB2018, R&D Systems), anti-OCT4 (sc-8628, Santa Cruz), anti-KLF4 (AF3158, R&D Systems), anti-MYC (AF3696, R&D Systems), anti-histone H3 (ab1791, Abcam), anti-ACTIN (A5316, Sigma), and anti-GAPDH (G8795, Sigma). DAPI was purchased from Sigma (D9542).

iPSCs or EBs were seeded on 0.1% gelatin-coated glass coverslips in 24-well culture dishes and fixed with 4% paraformaldehyde (PFA) in PBS for 15 or 20 min at room temperature, washed twice with PBS, and then permeabilized with 0.1% Triton X-100 in PBS for 5 min. After 1 hr in blocking solution (5% FBS in PBS), iPSCs were incubated with primary antibodies directed against Oct4 (1:200, Abcam, ab18976), Sox2 (1:200, Abcam, ab97959), Nanog (1:100, Novus Biologicals, NB100-58842) or SSEA-1 (1:100, Cell Signaling, MC480), and EBs with antibodies directed against AFP, Brachyury, or Nestin followed by repeated washing and incubation with secondary antibodies (goat anti-mouse immunoglobulin G (IgG) (H+L) or goat anti-rabbit IgG (H+L), Invitrogen). Nuclei were counterstained with DAPI and mounted with ProLong Gold antifade reagent (Life Technologies).