To test the interactions of ZMYM2 with LSD1 and CHD4, 2 × 15 cm dishes of confluent ESCs were harvested, and nuclear extracts were prepared as described (Ding et al., 2015 (link)). For immunoprecipitation, nuclear extracts were incubated with 4 μg ZMYM2 (Abcam, ab30783), LSD1 (Abcam, ab17721), or IgG (Millipore, PP64) antibodies and then incubated with protein G-Agarose beads (#11243233001, Roche) overnight at 4 °C. The immunoprecipitates were washed five times with wash buffer (50 mM HEPES, pH 7.9, 180 mM NaCl, 0.1% NP-40, 0.2 mM EDTA) containing 0.2 mM PMSF, protease inhibitor cocktail and 0.5 mM DTT. Proteins were eluted from the beads by boiling in wash buffer and 4X SDS loading buffer. Western blotting with the following primary antibodies were performed: ZMYM2 (Abcam, ab30783), LSD1 (Abcam, ab17721), CHD4 (Abcam, ab70469), HDAC1 (Bethyl, A300–713A), HDAC2 (Bethyl, A300–705A-1).
For FLAG IP, nuclear extracts were prepared from Zmym2-3xFLAG knockin ESCs and wildtype ESCs and incubated with 50 μl of α-FLAG-agarose beads (M2, Sigma) for 3 hrs. The immunoprecipitates were washed five times with wash buffer, eluted from the beads by boiling in wash buffer and 4X SDS loading buffer, and separated by SDS-PAGE. Western blotting was performed using the following primary antibodies: ZMYM2 (Abcam, ab30783), LSD1 (Abcam, ab17721), CHD4 (Abcam, ab70469) and HDAC2 (Bethyl, A300–705A-1).
For FLAG IP, nuclear extracts were prepared from Zmym2-3xFLAG knockin ESCs and wildtype ESCs and incubated with 50 μl of α-FLAG-agarose beads (M2, Sigma) for 3 hrs. The immunoprecipitates were washed five times with wash buffer, eluted from the beads by boiling in wash buffer and 4X SDS loading buffer, and separated by SDS-PAGE. Western blotting was performed using the following primary antibodies: ZMYM2 (Abcam, ab30783), LSD1 (Abcam, ab17721), CHD4 (Abcam, ab70469) and HDAC2 (Bethyl, A300–705A-1).