Fig. 3
In vitro characterization of the response of D2HGlo to L-2-HG. (A) IDH1-R132H catalyzes the conversion of α-ketoglutarate to D-2-HG. L-2-HG is produced by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH). (B) Representative in vitro D-2-HG and L-2-HG binding curves for purified D2HGlo. The average Kd’ ± standard deviation is shown for D-2-HG (blue) and L-2-HG (red) and represents 3–7 independent experiments. (C) The percent increase in FRET ratio above baseline levels in response to D-2-HG and L-2-HG at 1 µM (left) and 10 µM (right). The average ± standard deviation for each condition is shown for three independent experiments. Statistical analysis was performed using an unpaired t-test (****, P < 0.0001). (D) Representative in vitro D-2-HG binding curves for purified D2HGlo in a background of low concentrations of L-2-HG (1.5–5.5 µM, top) or high background concentrations of L-2-HG (15–55 µM, bottom). (E) The in vitro dynamic range of D2HGlo determined from three independent D-2-HG titrations in the presence of background L-2-HG ranging from 1.5–55 µM. (F) The Kd’ of D2HGlo for D-2-HG from three independent experiments performed in the presence of L-2-HG at concentrations ranging from 1.5–55 µM