Immunoprecipitation and immunoblotting

Cells were transiently transfected with the appropriated constructs and harvested 24 h later. Lysate preparation and subsequent immunoprecipitation were performed as described previously with minor changes (Tardat et al, 2015). Briefly, the lysates were cleared by centrifugation before pre‐clearing with 50 μl of mouse IgG agarose beads (Sigma) for at least 1 h at 4°C with rotation. After centrifugation, 25 μl of mouse anti‐Flag M2 agarose beads (Sigma) were added to the supernatant and allowed to precipitate overnight at 4°C with rotation. Beads were pelleted by centrifugation, washed three times, and resuspended in SDS buffer. For the analysis of immunoprecipitation, the proteins were separated by SDS–PAGE and detected with the following antibodies: anti‐Flag (Sigma, Cat#F1804), anti‐GFP (Roche, Cat#11814460001), anti‐Histone H3.3 (Millipore, Cat#09‐838), anti‐H3K27me3 (Cell Signaling, Cat#9733), anti‐Myc Tag (Abcam, Cat#Ab9132), and anti‐RNF2 (Active Motif, Cat#39663). For immunoblots, anti‐αTubulin (Sigma, Cat#T9026) was used as a loading control.