FIGURE 2.

HDAC8 deacetylates H3K27 in vitro. A, immunoprecipitated endogenous HDAC8 was prepared from wild-type RAW264.7 cells by immunoprecipitation using anti-HDAC8. Beads only or HDAC8 immunocomplex was incubated with histones purified from wild-type RAW264.7 cells in HDAC assay buffer in the presence or absence of PCI (20 nm) at 37 °C for 60 min. H3K27Ac levels were analyzed by Western blots (left panel). B, similarly, HDAC8-EGFP complexes were prepared from RAW264.7 cells stably transfected with pEGFP-HDAC8 using EGFP antibody. HDAC8-EGFP immune complexes were incubated with histones for the time indicated, and levels of H3K27Ac were analyzed by Western blot. Immunoblot for HDAC8-EGFP was used to visualize the immunoprecipitated HDAC8-EGFP. Histones used for substrates were visualized by Ponceau S staining. Images shown are representative results of three independent experiments. Band intensities compared with loading controls were analyzed by the ImageJ program (National Institutes of Health) and are expressed as means ± S.D. (n = 3); Student's t test; *, p < 0.05.