Immunoprecipitation of FLAG- and HA-tagged Proteins

We followed the protocol that we used recently.⁴⁵ (link) The plasmids of Lenti-FLAG-BRD4-GFP, Lenti-HA-EED, Lenti-FLAG-EED (and FLAG-tagged truncated EED constructs), Lenti-HA-STAT3-GFP, and Lenti-HA-EZH2-GFP were constructed and each was used to transfect HEK293 cells (Addgene, catalog no. 60360) in the presence of the JetPRIME transfection reagent. Cells were lysed on ice for 30 min in Pierce IP Lysis Buffer (Thermo Fisher Scientific, catalog no. 87788) containing Halt Protease Inhibitor Cocktail (87785; Thermo Fisher Scientific), and then centrifuged at 13,200 × g for 15 min at 4°C. The supernatant was incubated with 50 μL of Pierce Anti-DYKDDDDK Magnetic Agarose beads (A36797; Thermo Fisher Scientific) at 4°C overnight. The beads were washed 3× with cold PBS buffer and then incubated in 0.1 M glycine (pH 2.8) for 10 min at room temperature with frequent vortex to elute the immunoprecipitates. The eluate was neutralized with 1 M Tris-HCl, pH 8.5 (15 μL per 100 μL of eluate) and briefly heated at 95°C before its use for SDS-PAGE and western blot analysis.