Fig 1: Thermal Shift Assay. Effect of AG-120 on the thermal stability of IDH1wt (A) and IDH1R132H (C), assessed by Western blotting using a pan-IDH1 or IDH1R132H-specific antibody, respectively. Representative experiments are shown. Abbreviations: D = DMSO (vehicle control); A = 18 µM AG-120. (B, D) Densitometric analysis of immunoblots shown in (A) and (C), respectively. Band intensities corresponding to IDH1wt or IDH1R132H were quantified by densitometry and expressed as percentages relative to the intensity measured at 40 °C. The resulting values were plotted against temperature and fitted using a four-parameter sigmoidal model. The midpoint of each curve, representing the temperature at which 50% of the initial band intensity remained, was defined as the apparent aggregation temperature (Tagg). Tagg values for the representative experiments are shown as follows: IDH1wt, vehicle = 48.9 °C and 18 µM AG-120 = 53.3 °C; IDH1R132H: vehicle = 48.6 °C and 18 µM AG-120 = 55.1 °C
Fig 2: PAGE autoradiography and Western blot. Cytosolic fractions from IDH1wt-U251 or IDH1R132H-U251 cells were separated by native polyacrylamide gel electrophoresis 90 min after incubation with [18F]AG-120, followed by autoradiographic analysis of the gel. Subsequently, proteins were transferred to a PVDF membrane and the respective enzymes were detected by fluorescence immunostaining (3 independent experiments). (A) Autoradiography of a representative experiment (working concentration [18F]AG-120 = 1 nM; protein concentration: IDH1wt-U251 cytosol = 3.5 mg/mL, IDH1R132H-U251 cytosol = 3.4 mg/mL). (B) Corresponding Western blot (anti-h-IDH1 = Dianova W-09, 1:1000; anti-h-IDH1R132H = Dianova H-09, 1:1000)
Supplier Page from Abcam for Recombinant human IDH1 protein (His tag N-Terminus)