Figure 7.

Phosphorylation profile of p53 acetylation mutant proteins. (A) Cell extracts derived from tetracycline-induced H1299 cells expressing p53, p53Q320, or p53Q373 were subjected immunoblot with antibodies recognizing total or S15-phosphorylated p53. p53 levels were equalized among the different cell lines (bottom), so that similar amounts of each p53 acetylation mutant was probed with anti-phosphorylation–specific antibodies. S46 or S392 phosphorylation was detected via immunoprecipitation with a p53 polyclonal antibody, followed by immunoblot with anti-p53 antibodies directed against phospho-S46 or -S392. Fold induction for each phosphorylation site was determined by densitometry by using Fluor Chem 3.04A software, whereby cells expressing native p53 were attributed an arbitrary value of 1 and signals were normalized for total p53 levels. (B) WT p53, p53Q320, and p53Q373 were purified from baculovirus-infected cells and immunoprecipitated (IP) with different antibodies, each recognizing a different epitope, at the concentrations indicated at the top of each panel. The products of these immunoprecipitations were subjected to immunoblot (IB). The combination of antibodies used is indicated at the left side of each panel. (C) p53 mutants purified from baculovirus-infected cells were visualized via Coomassie blue staining (1) or by direct immunoblot with the DO-1 antibody (2).