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. 2015 Jul 24;5:12485. doi: 10.1038/srep12485

Figure 2. Transcriptional Activation of MEIS1 by RE-IIBP-Mediated H3K79 Methylation.

Figure 2

(a) and (b), MEIS1 mRNA levels were analyzed using real-time PCR in RE-IIBP transiently transfected 293T cells and stable RE-IIBP knocked-down 293T cells using two independent shRNAs (left). Cells were lysed and immunoblotted with anti-MEIS1 and anti-RE-IIBP antibodies. β-actin was used as a loading control (right). (c), 293T cells were transfected with pGL3-MEIS1 promoter, increasing concentrations of GAL4-RE-IIBP constructs, GAL4-RE-IIBP R477A, shCTL, shRE-IIBP#1, and shRE-IIBP#2. Following transfection, cell extracts were assayed for luciferase activity. Luciferase activity was normalized to that of β-galactosidase. Results are shown as means ± SDs n = 3; **p < 0.01. (d), MMSET stably knocked-down K562 were analyzed by Western blot. (e), Schematic diagram of primer pairs in ChIP analysis (Top). 293T cells were transfected with HA-RE-IIBP. Following transfection, ChIP analysis was performed employing anti-IgG, anti-HA, and anti-H3K79me2 antibodies. The immunoprecipitated DNA fragments were analyzed by RT-PCR from the proximal (Top) and distal promoter regions of MEIS1 (Bottom).