FIG. 4.

Deacetylation of MEF2D by HDAC3. (A) The expression construct for hHDAC3i (shRNA specific to human HDAC3) (lane 1) or the corresponding empty vector (lane 2) was transfected into HEK293 cells. Extracts were prepared for immunoblotting with anti-HDAC3 (αHDAC3) and anti-MEF2D (αMEF2D) antibodies. (B) The Flag-MEF2D expression plasmid was transfected into HEK293 cells along with the hHDAC3i expression construct or the corresponding empty vector as indicated. Before harvesting, cells were treated with 3 μM trichostatin A for 1.5 h (+) or not treated with 3 μM TSA (−). Extracts were prepared in buffer K supplemented with the inhibitor for affinity purification on M2 agarose, and bound proteins were eluted for Western blotting (WB) with anti-acetyl lysine (αAc-K) or anti-Flag (αFlag) antibody. (C) The Flag-MEF2D expression plasmid was transfected into HEK293 cells with or without plasmids expressing Flag-tagged HDAC3 and mutant H134Q. Before harvesting, cells were treated with 3 μM TSA for 1 h. Extracts were prepared in buffer K with the inhibitor for immunoprecipitation on M2 agarose, and bound proteins were eluted for immunoblotting with anti-acetyl lysine (αAc-K) or anti-Flag (αFlag) antibody. (D) Flag-HDAC proteins, expressed in and affinity purified from HEK293 cells, were used for in vitro deacetylation of Flag-MEF2D, which was expressed separately in and affinity purified from HEK293 cells. Before harvesting, the cells expressing Flag-MEF2D were treated with 3 μM TSA for 6 h. Flag-MEF2D was then affinity purified on M2 agarose using buffer K containing 3 μM TSA. (E) Same as panel C except that the effects of HDAC4 and CaMKIV-ca (a constitutively active form) were analyzed. (F and G) Flag-tagged HDAC1 and HDAC3 proteins, affinity purified from HEK293 cells, were used for deacetylation of Flag-MEF2D, acetylated in vitro with [¹⁴C]acetyl-CoA by Flag-PCAF (F) or Flag-p300 (G). Flag-tagged MEF2D, PCAF, and p300 proteins were affinity purified from Sf9 cells. (H) Expression plasmids for Flag-PCAF and HA-MEF2D were transfected into HEK293 cells individually or in combination. Extracts were prepared for immunoprecipitation (IP) and immunoblotting or Western blotting (WB) with anti-Flag or anti-HA antibody as in Fig. 2A. (I) HEK293 cells were transfected with plasmids expressing the indicated proteins. Before harvesting, cells were treated with 3 μM TSA for 1.5 h. Extracts were prepared in buffer K with the inhibitor for immunoprecipitation on M2 agarose, and bound proteins were eluted for immunoblotting with anti-acetyl lysine or anti-Flag antibody.