FIG 5.

MDM2 AD and RING domains bind to p53 core domain. (a, b) Beads loaded with GST-p53 and GST-R175H deletion mutants were incubated with H1299 lysate containing MDM2GP to form p53/MDM2GP complexes that were then cleaved with PreScission. The release of MDM2 fragments from the p53 complex was detected by Western blotting. Numbers above lanes show p53 residues present in each construct. (c) Diagram of MDM2 AD and RING binding to p53 core domain. (d) H1299 cells cotransfected with MDM2GP and p53 were treated with 20 μM 17-AAG for 5 h. MDM2GP-p53 complex was captured on Pab421 beads and cleaved by PreScission. The release of MDM2 fragments from p53 complex was detected by Western blotting. (e) SJSA cells were cultured in medium containing 17-AAG (50 μM) for 24 h. The turnover of MDM2 and p53 was analyzed by Western blotting after treatment with cycloheximide (CHX; 100 μg/ml).