Ctbp1

Whole cell lysates were prepared by lysing cells with RIPA buffer (0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 25 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA) supplemented with a protease inhibitor mixture and PhosSTOP (Roche). Histones were prepared by acid extraction as previously described (Choi et al., 2016 (link)). Proteins were quantified with Bradford assay (Bio-Rad). Equal amounts of proteins were separated with SDS-PAGE and transferred to nitrocellulose membranes. To visualize equal protein loading, blots were stained with Ponceau S. Blots were incubated in 5% non-fat milk in TBST, probed with primary antibodies to HA (1:1,000, Cell Signaling Technology, Cat# 2367), CtBP2 (1:1000, BD Biosciences, Cat# 612044, RRID:AB_399431), Mek1/2 (1:1,000, Cell Signaling Technology, Cat# 9122), alpha-tubulin (Sigma, Cat# T5168), MLL1 (1:1,000, Cell Signaling Technology, Cat# 14197), CtBP1 (1:1,000, BD Biosciences, Cat# 612042, RRID:AB_399431), Myc tag (1:1,000, Cell Signaling Technology, Cat# 2276), V5 (1:1,000, Invitrogen, Cat# R950-25), Histone H3K4me3 (1:1,000, Cell Signaling Technology, Cat# 9751, RRID:AB_2616028), and Histone 3 (1:1,000, Cell Signaling Technology, Cat# 5427) and then were incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized by enhanced chemical luminescence (Cytiva or Pierce).

Cells were lysed in Sarin buffer (20 mM tris-HCl, 138 mM NaCl, 10 mM EDTA, 50 mM NaF, 1% Triton, and 10% glycerol), which was supplemented with protease inhibitors SigmaFast and Na3VO4 and reducing agent dithiothreitol (DTT) (all chemicals purchased from Sigma- Aldrich). Protein content was measured by Pierce BCA protein Assay (Thermo Fisher Scientific, catalog no. 23227). Protein (40 μg) was loaded on a 4 to 12% Bis-Tris polyacrylamide gel (Thermo Fisher Scientific, catalog no. NP0321) and ran in a Mini Gel Tank (Thermo Fisher Scientific) in 1X Mops buffer (Thermo Fisher Scientific catalog no. NP001). Proteins were semi-dry blotted onto a 0.2 μM nitrocellulose membrane (Sigma-Aldrich, catalog no. GE10600001), and protein levels were detected by specific antibodies directed against Human EVI1 (Cell Signaling Technology, catalog no. 2265), CTBP1 (BD, catalog no. 612042), CTBP2 612044 (BD, 612044), V5 tag (Life Technologies, catalog no. R96025), FLAG tag (Sigma-Aldrich, catalog no. F3165), β-actin (Sigma-Aldrich, catalog no. no A5441), and hemagglutinin tag (Santa Cruz Biotechnology, catalog no. no Sc-805). Proteins were visualized using the Odyssey infrared imaging system (LI-COR Biosciences).

hESCs cultured on γ-irradiated MEFs on chamber slides were fixed in 4% paraformaldehyde for 15 min. Non-specific antibody binding was blocked with 10% fetal calf serum and, where necessary, cells were permeabilized with 0.2% Triton X-100 for 30 min. Cells were incubated with primary antibodies diluted in 0.6% BSA for 90 min. Primary antibodies used were CTBP1 (BD Biosciences; 612042) 1:200, CTBP2 (BD Biosciences; 612044) 1:250, OCT4 (Santa Cruz; sc-5279) 1:100, SOX2 (Cell Signaling Technology; D6D9) 1:200, NANOG (Abcam; ab109250) 1:100, and TRA-1-60 (Santa Cruz; sc-21705) 1:100. Cells were incubated with secondary antibody, goat anti-mouse IgG-fluorescein isothiocyanate (FITC) (Sigma; F2012) 1:100, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen; A-11008) 1:700, or goat anti-mouse IgM-FITC (Sigma; F9259) 1:200, for 60 min. Cells were mounted in VECTASHIELD with DAPI (Vector Laboratories) and visualized using a Zeiss fluorescence microscope and Axiovision imaging software.