A nanog

For MSL1 antibody production, a GST-mMSL1 fusion protein (C-terminal, residues 254–616) was used to immunize rabbits; the final bleed was used in experiments. Antibody specificity was verified with IP and MSL1-specific RNAi followed by Western blot analysis and ChIP assay. We used several commercial antibodies: a-KANSL1 (PAB20355; Abnova, Taiwan), a-KANSL3 (HPA035018; Sigma), a-MCRS1 (11362-1-AP; Proteintech, Chicago, IL), a-MOF (A3000992A; BETHYL Montgomery, TX), a-MSL2 (HPA003413; Sigma), a-NANOG (A300-397A; BETHYL), a-OCT3/4 (sc-5279; Santa-Cruz Dallas, TX), a-REX1 (Ab28141; Abcam, England), a-ESRRB (PP-H6705-00; Perseus Proteomics, Japan), a-KLF4 (Ab72543; Abcam), a-SOX2 (AF2018; R&D Systems, Minneapolis, MN), a-YY1 (A302-779A; BETHYL), a-RNF12/RILM (16121-1-AP; Proteintech) a-GAPDH (A300-639A; BETHYL), a-NESTIN (Ab93666; Abcam), a-CTCF (Ab70303; Abcam), a-H3 (Ab1791; Abcam), a-H4 (Ab10158; Abcam), a-H4K16ac (07-329; Millipore, Billerica, MA).

Cells were lysed with lysis buffer comprising 50 mM Tris pH 8.0, 150 mM NaCl supplemented with fresh 0.5% NP-40, 0.5 mM DTT, 1 × protease inhibitors cocktail (Roche, Switzerland) and 1.3 μl of Benzonase (Novagen, Germany) (1 hr, 4°C). Samples were prepared by boiling 40 μg of total protein extract with Laemmli buffer (Life Technologies, Cambridge, USA) . Samples were analysed using Bolt 10% Bis-Tris +SDS PAGE (Life Technologies, Cambridge, USA) and electroblotted onto 0.2 µm pore Whatman-Protran nitrocellulose membranes (Capitol Scientific, Austin, USA) in transfer buffer comprising 25 mM Tris/0.21M glycine/20% methanol. Membranes were blocked using 5% (w/v) low-fat milk in 0.01% (v/v) Tween-20/PBS (PBSTw) before incubating with primary antibody in blocking solution . Membranes were washed with PBSTw before incubating with donkey-raised secondary antibodies conjugated with IRDye 800CW (1:10000; LI-COR 926–32213, Lincoln, USA) and HRP-conjugated α-βActin (1:10000; Abcam ab20272, UK) antibody. HRP-staining was developed using a Super-signal West Pico kit (Pierce, Cambridge, USA) before imaging the membranes using LI-COR Odyssey Fc imager. The primary antibodies used were: α-Sox2 (1:1000; Abcam ab92494, UK) and α-Nanog (1:2000; Bethyl Laboratories A300-397A, Montgomery, USA).