Fig. 5: Phosphorylation of Ezh2 by Akt dissociates Ezh2 from the promoter regions of major TF loci
. a WT Pmel-1 cells were stimulated with anti-CD3/CD28 Ab + IL-2. Immunoblot analysis of Pmel-1 cells stimulated in vitro for 3 days, 5 days, and 7 days, probed with Abs against Ezh2 and phosphorylated Ezh2S21. Unstimulated TN were used as control. b–d Immunoblot analysis of Pmel-1 CD8+ T cells stimulated in vitro for 7 days, with or without treatment of MK2206, probed with indicated Abs. e–g WT Pmel-1 cells were cultured with anti-CD3/CD28 Ab + IL-2, with or without treatment of PI103, MK2206 or rapamycin for 7 days. Real-time RT-PCR analysis of Ezh2-targeted genes (e). ChIP analysis of cultured Pmel-1 cells treated with PI103, or MK2206. Graphs show the deposition of Ezh2 (f) and H3K27me3 (g) at the promoter regions of Id3, Id2, Prdm1, and Eomes. h–k WT Pmel-1 cells were stimulated with anti-CD3/CD28 Ab for 36 h, followed by infection with MigR1 retrovirus (GFP) or MigR1 retrovirus encoding flag-tagged Ezh2, or Ezh2S21A. Pmel-1 cells were collected at 7 days after culture. Immunoblot analysis of GFP and Ezh2S21A Pmel-1 cells, probed with indicated Abs (h). Real-time RT-PCR analysis of their expression of major TFs (i). ChIP analysis shows the deposition of Ezh2 (j) and H3K27me3 (k) at the promoter regions of these major TFs loci. *p < 0.05, **p < 0.01, and ***p < 0.001 (two-tailed unpaired t test). The data are representatives of three independent experiments (a–d), or two experiments (e–k; mean ± SD)
