Ab7028

Co-immunoprecipitation experiments was also performed as described (Whyte et al., 2012 (link)). Briefly, undifferentiated F9 ECCs and ESCs were washed and harvested in cold 1X PBS. Cellular proteins were extracted using TNEN250 lysis buffer (50 mM Tris pH 7.5, 5 mM EDTA, 250 mM NaCl, 0.1% NP-40) complemented with protease inhibitors at 4°C with rotation for 30 min. Complexes were then immunoprecipitated overnight at 4^°C with rotation by incubating the supernatant solution supplemented with two volumes of TNENG (50 mM Tris pH 7.5, 5 mM EDTA, 100 mM NaCl, 0.1% NP-40,10% glycerol) with Dynabeads® M280 (Life Technologies, 11203D) bound to 5 ug of antibody. Beads were washed withTNEN125 (50 mM Tris pH 7.5, 5 mM EDTA, 125 mM NaCl, 0.1% NP-40) and samples were eluted by boiling for 10 min in Laemmli’s loading buffer containing 100 mM DTT. Western blots were performed with NuPAGE4%–12% Tris-Bisgels. Antibodies used included: Lsd1 (abcam, ab17721), HDAC1 (abcam, ab7028), Mi-2b (abcam, ab72418).

For co-immunoprecipitation (Co-IP), the nuclear extract was prepared according to manufacturer’s protocol (Active Motif, 40010) except that DNase was added for the release of chromatin-associated proteins. The Co-IP was performed using 1:1 mix of Dyna- beads Protein A (Life Technologies, 1002D) and Dyna-beads Protein G (Life Technologies, 1004D), and conjugated with 5 mg antibody and with 50 mg of nuclear extract according to the manufacturer’s protocol. Antibodies used include: anti-Oct4 (Abcam, ab181557), anti-CHD4 (Abcam, ab72418), anti-HDAC1 (Abcam, ab7028), anti-Lsd1 (Abcam, ab17721) and anti-HBO1 (Abcam, ab124993).
Pull down assays were performed using 1 mg of GST-Lsd1 (Sigma, SRP0122) incubated with 1 mg of recombinant Oct4 (abcam, ab134876) and Glutathione Sepharose 4B (GE healthcare, 17–0756-01) resin in the binding buffer (50 mM Tris pH 8.5, 50 mM KCl, 5 mM MgCl, 0.5% BSA, and 5% glycerol, complemented with a cocktail of protease inhibitors) overnight at 4°C with gentle agitation. The resin was washed twice with binding buffer and proteins were eluted using the elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8) according to the manufacturer’s instructions. Eluate and input were loaded onto a 10% SDS-PAGE gels and blots were probed using anti-Lsd1 (Abcam, ab17721) and anti-Oct4 (Santa Cruz, sc-8628) antibodies.