Ab181557

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.