Figure 2.

IDH1-R132H is required for pH sensitive d-2-hydroxyglutarate production in mammalian cells. (A) Western blot analysis shows equal expression of FLAG-tagged IDH1-WT and IDH1-R132H when stably expressed in NIH-3T3 cells (3T3). Blots shown are duplicate blots with equal loading of 5 μg of total protein in each lane followed by anti-actin and anti-FLAG antibody detection. (B) Intracellular pH (pHi) can be controlled in 3T3 cells using a small molecule inhibitor of NHE1 (EIPA, 1 μM) to decrease pHi and ammonium chloride (NH₄Cl, 5 mM) to increase pHi (see Methods for details). (C) 3T3 cells with pHi manipulated as in panel B were assayed for levels of the oncometabolite D-2HG and (D) AKG (see Methods for details). Data in panels B and C are from four biological replicates and shown as box and whisker plots (Tukey display). Data in panel D are from three biological replicates and shown as scatter dot plots (mean ± standard error of the mean). For panels B–D, significance was determined by a two-way analysis of variance with Tukey multiple-comparison correction (*p < 0.05, **p < 0.01, and ***p < 0.001). For panel D, there were no statistically significant differences across the data set.