FIGURE 4.

Lys-289 and Lys-309 are the major sites in SREBP-1c acetylated by p300. A, the experimental protocol for MS/MS analysis is outlined. B, purified GST or GST-SREBP-1c was incubated with p300, and proteins were separated using SDS-PAGE followed by Western analysis. C, MS/MS analysis of acetylated SREBP-1c is shown, which identifies Lys-289 as a site acetylated by p300. D, a schematic diagram of the acetylation sites in SREBP-1c. E, alignments of the SREBP-1c region containing Lys-289 or Lys-309 from different vertebrates. F, outline of experimental protocols. G, COS-1 cells were transfected with plasmids as indicated, and acetylation assays were performed as described in the legend to Fig. 1. H, band intensities of acetylated SREBP-1c were quantified, and the values from wild type samples were set to 1. Consistent results were observed from two additional assays. I, in vitro acetylation assays were performed as described under “Experimental Procedures.” Acetylated FLAG-SREBP-1c was detected by Western analysis. IP, immunoprecipitation; WB, Western blot. Error bars, S.E.