Fig. 5. EED and BMI1 engage in separate protein complexes. Immunoprecipitation of EED (left column) from E12.5 trunk identified three isoforms of approximately 50 and 75 kDA. Note the absence of the EED isoforms in the input lane, indicating low levels of EED expression. EED co-immunoprecipitated with EZH2 and dimethylated H3-K27, which presented a molecular weight of approximately 100 and 17 kDA, respectively. Immunoprecipitation of BMI1 (right column) from E12.5 trunk revealed three isoforms in the 39-41 kDA range. A fourth band, slightly larger than the triplet, was occasionally detected in mock immunoprecipitation without the BMI1 antibody and, hence, could not be confirmed as a BMI1 isoform. RING1B co-immunoprecipitated with BMI1 as a band of approximately 38 kDa. Note that reciprocal co-immunoprecipitation did not detect EED and BMI1 in a common protein complex, and trimethylated H3-K27 did not pull down with either complex. YY1, as a band of 49 kDA, co-immunoprecipitated with both EED and BMI1. IP, immunoprecipitation; beads, mock immunoprecipitation without antibody; input, 2 µg protein lysate; H3M2K27, dimethylated histone 3-K27, H3M3K27, trimethylated histone 3-K27.