Figure 4.

PTH stimulates p300 HAT activity, which requires protein synthesis, and also stimulates MMP-13 promoter-bound p300. A, HAT assays were performed as described in Materials and Methods, using lysates (1 mg protein) from UMR cells, control or treated with rat PTH (peptide 1–34, 10⁻⁸ m). Whole-cell lysates were immunoprecipitated with anti-p300 antibodies, mouse IgG, anti-CBP-NT antibodies, or rabbit IgG. Results are expressed in picomoles/mg protein/h and represent means ± sem from triplicate samples. B, PTH stimulation of p300 HAT activity requires protein synthesis. HAT assays were performed on lysates from UMR cells treated with cycloheximide (30 μg/ml) at the time of PTH addition and immunoprecipitated with anti-p300 antibodies or mouse IgG. C, ChIP assays using UMR cells, untreated or treated with rat PTH (peptide 1–34, 10⁻⁸ m) for various times and immunoprecipitated with anti-p300 antibody. Quantitative real-time PCR was done using MMP-13 promoter primers, −204/−34. Error bars represent the sd of three independent experiments. †, # , and *, P < 0.05, P < 0.005, and P < 0.001 compared with vehicle-treated control, respectively. Chx, Cycloheximide; ctrl, control.