FIG. 2.

Mapping the HDAC3-binding site on MEF2. (A) Flag-tagged MEF2D and deletion mutant 1-86 were expressed with HA-HDAC3 in HEK293 cells as specified. Extracts were prepared in buffer K for purification on M2 agarose, and bound proteins were eluted with Flag peptide for Western blotting (WB) with anti-HA (αHA) or anti-Flag (αFlag) antibody. (B) HA-tagged MEF2C mutants 1-116 (HA-1-116) and 1-178 (HA-1-178) were expressed with Flag-HDAC3 in HEK293 cells as indicated. Extracts were prepared and analyzed as described above for panel A. IP, immunoprecipitation. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel. (C) Extracts from bacteria expressing MEF2C fragments fused to MBP were incubated with amylose resin to immobilize the fusion proteins and pull down HA-HDAC3, synthesized in vitro in the presence of [³⁵S]methionine. The bound proteins were resolved by SDS-PAGE for Coomassie blue staining (bottom) and autoradiography (top). Extracts with MBP fused to a small portion of β-galactosidase were used as a negative control (lane 2). About 20% of the total HA-HDAC3 protein used per binding reaction was analyzed on the input lane. (D) Extracts from Sf9 insect cells expressing Flag-HDAC3 were incubated with M2 agarose to immobilize this fusion protein to pull down MEF2D or its deletion mutant 148-522, synthesized in vitro in the presence of [³⁵S]methionine. Plain Sf9 extracts were used as a negative control (lanes 2 and 5). About 20% of each protein used per binding reaction was analyzed on the input lanes. (E) HEK293 cells were transfected with expression constructs for Flag-MEF2D (2 μg) and HA-HDAC3 (6 μg) along with increasing amounts of the expression vector for HA-HDAC4 (0, 1, and 2 μg). Extracts were prepared and analyzed as described above for panel A.