Fig. 1. In vivo acetylation of MCM3. (A) The soluble protein fraction and the structure-bound protein fraction prepared from 5 × 10⁵ asynchronous cells were analyzed by 7.5% SDS–PAGE. The proteins were blotted to PVDF membrane. The membranes were incubated with indicated antibody, respectively. The bound antibody was detected by horseradish peroxidase-labeled secondary antibody and visualized by ECL PLUS kit. (B) HeLa cell extracts prepared from 1 × 10⁷ cells were incubated with protein A–Sepharose and with anti-MCM3 antibody or normal rabbit IgG (mock). After 1 h incubation, column-bound proteins were analyzed by SDS–PAGE (7.5%) and blotted to PVDF membrane. (C) MCM3/pcDNA3.1 plasmid or mock vector was transfected to 293T cells, respectively. After culture for 20 h the cells were harvested and resuspended in hypotonic buffer containing 1% NP40, 0.5 M NaCl, and 7 mM Na butyrate. Cell extracts were incubated with a TALON column and the column was washed extensively with same buffer. Bound proteins were extracted from the column with 100 mM imidazole and 7 mM Na butyrate in hypotonic buffer and analyzed by SDS–PAGE. The analyzed proteins were blotted to PVDF membrane.