(a) p53+/+ and p53−/− HCT116 cells, and U2OS cells transfected with p53 shRNA and control (ctrl) shRNA, were analyzed by RT-PCR with reverse transcription (top) and western blot (bottom).
(b, c) p53+/+ and p53−/− HCT116 cells were treated with or without 20 μM of PFTα for 24 h (b), or treated with or without Dox (2 μM) for 1 h, and then with or without cycloheximide (CHX, 20 μM) for 2 h (c). Cells were analyzed for G6PD activity (top) and protein expression (bottom). Data are means ± S.D. (n=3).
(d) H1299 cells were transfected with enhanced GFP-G6PD alone or together with increasing amounts of Flag-p53. Cell lysates were immunoprecipitated with an anti-Flag antibody and an isotype-matching control antibody (IgG). Immunoprecipitated proteins (IP) and 5% input were analyzed by western blot.
(e) p53+/+ HCT116 cells were treated with MG132 (20 μM), doxorubicin (DOX, 2 μM), or vehicle (DMSO). Cell lysates were incubated with anti-G6PD antibody or a control antibody (IgG). IP and input were analyzed by western blot.
(f) Left: Schematic representation of p53 and its deletion mutants. WT, wild-type; TA, transactivation domain; DBD, DNA-binding domain; CT, C-terminal region; TET, tetramerization domain; NR, negative regulation domain. The amino acids at the domain boundaries are indicated. Right: purified GST fusions of wild type and mutant p53 proteins were incubated separately with recombinant Flag-G6PD protein conjugated to beads. Beads-bound and input proteins were analyzed by western blot using anti-Flag (top) and Coomassie blue staining (bottom).
(g) Dissociation constant (KD) for p53 from immobilized G6PD was determined by surface plasmon resonance (BIAcore). A real-time graph of response units (RU) against time is shown. The on rate was 7.53 ± 1.59 × 103 M−1s−1, and the off rate was 1.30 ± 0.03 × 10−3 s−1.