Figure 3.

PML K487 is the major acetylation site and is critical for nuclear localization of PML in HeLa cells. (a) HeLa cells were transfected with HA-PML4 (wild-type, K487R, K515R, and K487/515R) and WCEs were analyzed by immunoprecipitation with an anti-HA antibody followed by immunoblotting with anti-acetyl-lysine and anti-HA antibodies. (b) HeLa cells were transfected with HA-PML4 (K515R) and Myc-SIRT1 (wild-type or H363Y mutant). WCEs were prepared and analyzed by immunoblotting with anti-HA and anti-Myc antibodies (upper panels). The WCEs were analyzed by immunoprecipitation with anti-HA antibody followed by immunoblotting with anti-acetyl-lysine and anti-HA antibodies (lower panels). (c) HeLa cells were transfected with HA-PML4 (wild-type, K487R, K515R, and K487/515R mutants). Cells were immunostained with anti-HA antibody and images were taken by fluorescence microscope. DAPI (4,6-diamidino-2-phenylindole) was used to indicate nuclei