Fig 6. Loss of in vitro HDAC activity of immuno-purified HDAC4c mutants due to inability to associate with endogenous SMRT-HDAC3 complex.

(A) In vitro HDAC assay and (B) co-immunoprecipitation assay for HDAC4c SRID mutant proteins immuno-purified from HEK293 cells. The HA-tagged versions of the indicated HDAC4c proteins were coexpressed with HDAC3 in HEK293 cells by transient transfection and purified from cell lysates (300 μg) with the use of agarose beads coupled with anti-HA-antibody. (A) The immune complex was subsequently subjected to in vitro HDAC assay using fluorescent-coupled Lys acetamide as a substrate. HeLa nuclear extract (1 μg) was used as the positive control. The p-values for all compared groups between wild-type and mutants are less than 0.0001. RFU: relative fluorescence units. (B) For co-immunoprecipitation assay, immunoprecipitates from 1 mg of whole cell lysate were prepared and analyzed for the presence of HA-HDAC4c and HDAC3 by immunoblot analysis.