FIG 3.

ADA3 exclusively interacts with SIRT1 among class I, II, and III deacetylases. (A) HEK293T cells were transfected with empty vector or various FLAG-tagged HDAC and SIRT constructs. Forty-eight hours after transfection, cell lysates were subjected to immunoprecipitation by M2 FLAG-agarose beads, eluted with 3×FLAG peptide, and immunoblotted with anti-ADA3 or FLAG-HRP antibody. (B) HEK293T cells were transfected either with empty vector or FLAG-ADA3. Forty-eight hours after transfection, cell lysates were subjected to immunoprecipitation by M2 FLAG-agarose beads, and immunoprecipitates were immunoblotted with anti-SIRT1 or FLAG-HRP antibody. (C) HEK293T cell lysates were subjected to immunoprecipitation using anti-SIRT1 antibody, and immunoprecipitates were analyzed by Western blotting using anti-ADA3–HRP and SIRT1 antibodies. (D) In vitro GST pulldown assay in which 300 ng recombinant SIRT1 was incubated with 1 μg glutathione-bound GST or GST-FLAG-ADA3, followed by immunoblotting with anti-SIRT1 antibody. (E) Lysates of HEK293T cells transfected with FLAG-SIRT1 wild type or its catalytically inactive mutant, H363Y, were immunoprecipitated as described for panel A, followed by immunoblotting with anti-ADA3 and FLAG-HRP antibodies. The values represent the ratios of signal intensities of ADA3 bands over that of total SIRT1 immunoprecipitated as calculated using ImageJ.