FIGURE 4.

HDAC9 deacetylates Lys¹¹⁶ of ATDC and alters ATDC functions. A, lysates were prepared from 293T cells transfected with plasmids that express wild type or mutated ATDC or vector alone. Proteins were separated by electrophoresis, transferred onto a membrane, and analyzed by Western blot. B, HeLa cells were co-transfected with plasmids that express FLAG-HDAC9, HA-p300, or vector. Endogenous ATDC Lys¹¹⁶ acetylation and the expression levels of different proteins were determined with direct Western blot or immunoprecipitations and then followed by Western blotting using the indicated antibodies. C, murine Hdac9^+/+ and Hdac9^−/− fibroblasts were transfected with HA-ATDC expression plasmids, and cell lysates were collected and analyzed by immunoprecipitation with an anti-HA antibody and Western blotting with the anti-acetyl-Lys¹¹⁶-ATDC antibody. The blot was stripped and reprobed with anti-HA to confirm equal HA-ATDC protein levels. D, U2OS cells were transfected with plasmids expressing the indicated proteins. Thirty-six h after transfection, cells were subjected to 10 Gy of irradiation. One h post-irradiation, cell lysates were prepared and analyzed by Western blots with the indicated antibodies. E, U2OS cells were co-transfected with a GFP expression plasmid together with either vector or plasmids expressing HA-ATDC or FLAG-HDAC9. Twenty-four h after transfection, cells were subjected to 10 Gy of irradiation. Sixteen h post-irradiation, cells were fixed briefly with ethanol and subjected to FACS analysis for cell cycle distribution. IP, immunoprecipitation; IB, immunoblot.