Quantitative IDH1 Enzyme Activity Assay

An in vitro enzyme activity assay for wild type IDH1 was performed as previously described (8 (link)). In brief, 20 ng purified IDH1 and YF variant proteins that were pre-treated with or without recombinant active tyrosine kinases in an in vitro kinase assay were added to 100 μl enzyme activity assay buffer (25mM Tris-HCl (pH7.5), 10 mM MgCl₂, 5 mM DTT) containing 0.5 mM NADP⁺ (Sigma-Aldrich) and 1 mM isocitric acid (Sigma-Aldrich). To determine the wild type IDH1 activity in cells, endogenous IDH1 from 1×10⁷ cells was bound to protein G-Sepharose 4 Fast Flow beads (Sigma-Aldrich) by immunoprecipitation using IDH1 antibody (CST). To test IDH1 activity in xenograft tumor tissues, around 2 mg of total tumor lysates were used. After immunoprecipitation, the IDH1-bound beads were washed 3 times and eluted using IDH1 peptide (CST). Then 10 ul of the supernatant was added to 100μl assay buffer (25mM Tris-HCl, pH7.5, 10 mM MgCl₂, 5 mM DTT) containing 0.5 mM NADP⁺ and 1 mM isocitric acid. IDH1 activity was measured by recording absorbance of 340 nm in kinetic mode every 20 seconds for 15 minutes using a SpectraMax Plus spectrophotometer (Molecular Devices).