Extended Data Fig. 4: Purification and activity of IDH2(R140Q) dimers with wild-type IDH2 or mutants Q316E or I319M in trans.
From: Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations
a, Schematic of experimental approach: 293T cells were co-transfected with HA-tagged IDH2(R140Q) plus Flag-tagged wild-type, Q316E or I319M. After 2 days, cells were lysed and enzyme complexes were purified by HA-immunoprecipitation. Reactions were performed with purified enzyme, NADPH, αKG and varying doses of AG-221 as detailed in Fig. 3. b–e, Purity and dimerization of HA-precipitated enzymes were assessed by denatured SDS–PAGE with Coomassie staining (b), denatured SDS–PAGE with western blotting (c), native PAGE with Coomassie staining (d), or native PAGE with western blotting for the indicated proteins (e). Separate membranes were used for western blots. f, In vitro enzyme assays measuring rate of NADPH consumption of IDH2 dimers purified as in b–e. Reactions contained purified enzyme (10 μg ml−1), NADPH (0.3 mM), αKG (5 mM) and AG-221 at indicated concentrations. Data are mean ± 95% confidence intervals for triplicate reactions. Results are representative of ≥3 (b–d, f) or 2 (e) independent experiments. For gel source data, see Supplementary Fig. 2.
