Extended Data Fig. 5: Effect of 2-HG on Mdm2-mediated ubiquitination of mutant p53.
From: Malic enzyme 2 maintains protein stability of mutant p53 through 2-hydroxyglutarate
a-c, In vitro p53 ubiquitination assay was performed using purified HA-p53R282W (a), HA-p53R273H (b) and HA-p53 (c) incubated with purified Flag-Mdm2, E1, E2 and/or ubiquitin (Ub) in the presence or absence of 5 mM 2-HG for 60 min. Mixtures were immunoprecipitated with anti-HA agarose, and analyzed by western blot. d, Purified ME2 was incubated with pyruvate or NADPH for 60 min at room temperature (RT) before incubating with purified HA-p53G266E, Flag-Mdm2, E1, E2 and/or ubiquitin (Ub) for another 60 min. Mixtures were then immunoprecipitated with anti-HA agarose and analyzed by western blot. e, Lysates from SF188 cells transfected with Ctrl or ME2 siRNA for 48 hours were collected after 6 hours of 10 μM MG132 treatment, and immunoprecipated with anti-p53 antibody. Immunoprecipitates and input were analyzed by western blot using anti-p53 (HRP) and anti-Ub antibodies respectively. f, MDA-MB-231 cells transfected with Ctrl or ME2 siRNA for 48 hours in the presence or absence of 5 mM 2-HG were collected after 6 hours of 10 μM MG132 treatment and subjected to immunoprecipitation using anti-p53 antibody. Immunoprecipitates and input were analyzed by western blot using anti-p53 (HRP) and anti-Ub antibodies respectively. g and h, also related to Fig. 4e, Recombinant Flag-tagged p53 mutants (p53G266E, p53R175H, p53R280K and p53R282W) and p53 were analyzed on SDS-PAGE followed by coomassie staining of the gels. i and j, Surface plasmon resonance (BIAcore) measurement of the interaction between 2-HG and purified p53R175H (i), p53R282W(i), or p53(j). The Graphs of equilibrium response units (RU) and compound concentrations are shown. The estimated Kd of p53R175H and 2-HG interaction is about 37 nM, and around 66 nM for that of p53R282W and 2-HG interaction. All immunoprecipitation and western blot experiments were repeated independently three times.
