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. 2013 Jun 28;288(32):23554–23564. doi: 10.1074/jbc.M113.482497

FIGURE 3.

FIGURE 3.

Expression and cellular localization of ESC, interaction between ESC and E(z), and methylation levels of histone H3 in brains of nondiapause- and diapause-destined pupae. A, shown is a Western blot analysis of Har-ESC transiently expressed in Sf9 cells. This is a schematic diagram of full-length ESC (ESC-FL), N-terminal ESC (ESC-N), and C-terminal ESC (ESC-C), which were used for transfection, respectively. NLS, nuclear location site, is in red; WD domain is in blue; full-length ESC-GFP at 1, 2, and 5 μg, N-terminal-GFP at 0.5 and 1 μg, and C-terminal-GFP at 1 and 2 μg transiently expressed in Sf9 cells. B, cellular localization of transiently expressed recombinant ESC. Hoechst 33342 staining shows the cell nuclei. Full-length ESC-GFP, N-terminal ESC-GFP, and C-terminal ESC-GFP were transfected into Sf9 cells; pIZ-V5-GFP (EGFP) was used as a control. Scale bar, 20 μm. C, ESC is physically associated with E(z). The HzAm1 cell extracts were immunoprecipitated (IP) with anti-ESC antibody, and the immunoblot (IB) was performed with anti-EZH2 antibody. Rabbit serum (IgG) served as a negative control. The 84-kDa band is E(z), and the 52-kDa band is IgG heavy chain. D, a Western blot analysis of H3K27me3, H3K4me3, and H3K27Ac detects methylation or acetylation of histone H3. The antibody against total H3 served as the loading control.