Fig. 1.

HDAC5-EGFP has deacetylation activity and associates in vivo with the N-CoR-HDAC3 complex and heterotrimeric PP2A enzyme. A, Schematic of EGFP-FLAG-tagged HDAC5 with functional domains: MEF2, myocyte enhancer factor 2 binding domain; NLS, nuclear localization signal; Deacetylase, deacetylase activity domain; NES, nuclear export signal. B, The HDAC5-EGFP localizes to both the nucleus and cytoplasm in the HEK293 cell line; HDAC5 (green, EGFP), nuclear periphery (red, Mab414 against nuclear pore complex proteins), nucleus (blue, DAPI); 100× oil-immersion lens; Bar, 5 μm. C, HDAC5-EGFP-FLAG has deacetylation activity. Deacetylation activity of HDAC5-EGFP and EGFP, isolated from HEK293 cell lines, was measured (n = 6, AFU±S.D.) using Fluor-de-Lys assay in the absence (-) or presence (+) of trichostatin A. D, HDAC5-EGFP associates with known interacting partners. Immunoaffinity purifications were performed via the EGFP tag, co-isolated proteins were resolved by one-dimensional SDS-PAGE, stained with Coomassie Blue, and analyzed by mass spectrometry. Red, known members of the N-CoR-HDAC3 corepressor complex; Blue, the three subunits of protein phosphatase 2A.