Differential phosphorylation and localization of PRC2 proteins in LSCs. Ezh2 is differentially phosphorylated at threonine 487 (T487) in FLB1 and FLA2 leukemias. (A) MS/MS spectrum of phosphopeptide-enriched FLB1 extracts showing the phosphosite on Ezh2 T487. (B) Ion chromatogram of the phosphopeptide ESSIIAPVPTEDVDpTPPR in nuclear extracts shows an ∼ 3-fold up-regulation in FLB1 cells. Mass spectrometry analyses were performed in triplicates. (C) Ezh2 primary protein structure and amino acid alignment of the region encompassing the identified T487 phosphorylated residue. The putative nuclear localization site (NLS, yellow box) is well conserved through evolution and in the mouse Ezh1 paralog. The phosphosite T487 is also well conserved but not present in Ezh1. Amino acid delimitation of Eed-binding domain (EBD), SANT, and SET domains are shown. (D) Differential localization of Ezh2 in FLB1 and FLA2 cells as shown by confocal microscopy using anti-Ezh2 antibody. Although Ezh2 is mainly nuclear in FLA2, it is predominantly cytoplasmic in FLB1 leukemic cells. (E) Western blot analysis of nuclear and cytosolic extracts from FLA2 and FLB1 leukemias for the core PRC2 complex proteins Eed, Ezh2, and Suz12 (representative results from replicate experiments using 4 different samples for each leukemia per experiment). Ezh2, Suz12, and specific isoforms of Eed are differentially localized in the nucleus and the cytoplasm. (F) Differences in nuclear and cytoplasmic abundance for Eed, Ezh2, and Suz12 in FLA2 and FLB1 cells. Levels were determined by analyzing the density of immunoblot signals using the Multi-Gauge imaging software from FUJIFilm. Graph represents the mean from 4 different samples for each leukemia.