MYCN physically interacts with Polycomb repressive complex 2 via EZH2. A, co-immunoprecipitation assay showing the interaction of endogenous EZH2 and MYCN in IMR-32 cells. Cell lysates were immunoprecipitated with the antibodies indicated on the top of the gel and subjected to Western blot analysis (WB) with the MYCN antibody. B, immobilized GST-MYCN polypeptides were incubated with equal amounts of the in vitro translated EZH2 protein, separated by SDS-PAGE, and probed with an anti-EZH2 antibody. The diagram in the bottom shows full-length MYCN protein in which the functional domains are indicated by different shades of gray. The MYCN segments cloned in the GST expression vector are in black, and numbers indicate amino acid positions. C, Western blot analysis showing equal expression of the wild type and mutant (mut; lacking amino acids 187–241) MYCN proteins in SH-SH5Y cells stably transfected with the different MYCN constructs. The MYCN-amplified cell line Kelly is used as a positive control for MYCN expression. D, co-immunoprecipitation analysis in SH-SY5Y cells transfected with the wild type or mutant MYCN constructs showing interaction of EZH2 wild type, but not mutant, MYCN protein. E, luciferase assay of SH-SY5Y cells expressing the wild type or mutant MYCN constructs, transiently transfected with the CLU promoter. Error bars indicate S.D., and the asterisk indicates statistical significance (Student's t test; p = 0.01, n = 4). F, Western blot analysis showing expression of CLU in SH-SY5Y cells stably transfected with the wild type or mutant MYCN plasmids. The bottom panel depicts the densitometric analysis of MYCN expression relative to actin.