Anti setdb1

Immunoblot analysis was performed using whole-cell extracts obtained by lysing the cell pellet with Triton buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 50 mM NaF, 1 mM EDTA 1 pH 8, 0.1% Triton) supplemented with proteases and phosphatases inhibitors. Proteins were resolved on an SDS-10% polyacrylamide gel and blotted onto a Hybond P PVDF membrane (G & E Healthcare). Membranes were blocked with PBST 5% non-fat dry milk, incubated with primary antibodies for 2 h at room temperature, washed and hybridized for 1 h at room temperature using the appropriate horseradish peroxidase-conjugated secondary antibody (rabbit and mouse; BioRad, Hercules, CA, USA). Detection was performed with the ECL chemiluminescence kit (Perkin Elmer, Waltham, MA, USA). The following antibodies were used: anti- β actin (Sigma AC15, dilution 1:50000), anti-SETDB1 (Thermo Scientific 5H6D4, diluition 1:1000), anti-FLAG rabbit (Sigma F7425, diluition 1:1000), anti-HA (Abcam ab130275, diluition 1:1000), anti-p63 BC4A4 mouse (Abcam ab735, diluition 1:200), anti-p63 rabbit (Abcam ab97865, diluition 1:200), anti-p63 4A4 mouse (Sigma P3737, diluition 1:500), anti-p63 3.1 mouse [54 (link), 63 (link)] (diluition 1:500), anti-p53 (Santa Cruz, diluition 1:1000), anti-H3K9me3 (Millipore, diluition 1:1000). Supplementary Figures S5–S10 show un-cropped images of western blots.

Whole cell lysates were extracted from 1 × 10⁷ ESCs using radioimmunoprecipitation assay buffer (RIPA buffer) with 1X PIC and phosphatase inhibitor phosSTOP™ (Roche). Protein concentrations were quantified using Quick Start™ Bradford Protein Assay (Bio-Rad) according to the manufacturer protocol. Lysates were subsequently resolved by 15% SDS-PAGE gel, wet-transferred onto PVDF membranes, and probed with 1:1000 anti-SETDB1 (ThermoFisher, PA5-30334), 1:1000 anti-CTCF (Active Motif, 61311), 1:1000 anti-FLAG (Sigma-Aldrich, F3165) or 1:20000 anti- β-Tubulin (Abcam, 6046) antibodies. Signals were developed with Clarity™ Western ECL Substrate (Bio-Rad) according to manufacturer’s protocol. Images were detected with ChemiDoc™ Touch Imaging System (Bio-Rad).