FIG. 5.
Interaction of HDAC3 with PCAF and p300. (A) Expression plasmids for Flag-PCAF and HA-HDAC3 were cotransfected into HEK293 cells as indicated. Extracts were prepared in buffer K for affinity purification on M2 agarose, and bound proteins were eluted for Western blotting (WB) with anti-HA (αHA) or anti-Flag (αFlag) antibody. (B) HEK293 cells were washed and lysed in buffer K in situ to prepare extracts for immunoprecipitation (IP) with anti-PCAF antibody (αPCAF) and immunoblotting with anti-HDAC3 or anti-PCAF antibody. Immunoblotting with anti-HDAC3 antibody was carried out as described in the legend to Fig. 1E. (C) Flag-HDAC3 and Myc-tagged p300 were expressed and purified as in described above for panel A by immunoblotting anti-Flag and anti-Myc monoclonal antibodies, respectively. (D) Extracts from Sf9 cells expressing Flag-p300 (lane 3) or Flag-PCAF (lane 4) were used to immobilize these fusion proteins on M2 agarose for incubation with HA-HDAC3, synthesized in vitro in the presence of [35S]methionine. Plain Sf9 extracts were used as a negative control (lane 2). About 20% of the total HA-HDAC3 protein used per binding reaction was analyzed on lane 1. (E and F) Flag-PCAF was expressed alone (E) or with GFP-HDAC3 (F) in HEK293 cells. Green fluorescence microscopy was used to detect GFP signals. Expression of Flag-PCAF was visualized by immunostaining with anti-Flag antibody and a Cy3-labeled secondary antibody. Hoechst 33258 was used to stain nuclei.