Figure 3

IDH1^R314C-BAPHIS expression in glioma cell lines leads to reduced forward activity compared to IDH^WT expression.

(A) Western blots showing expression of IDH1-BAPHIS in transiently transfected HEK293t and lentivirally transduced LN229 and U251 glioma cells. Blots were stained with anti-IDH1^PAN and anti-IDH1^R132H as indicated, or with anti-GAPDH as a loading control. Since the recombinant proteins are ~3 kDa larger than endogenous IDH1 they can be distinguished from the endogenous IDH1. Control LN229 and U251 were transduced with empty vector virus (EV). (B) Monitoring of NADPH formation after isocitrate/NADP⁺ addition to extracts from transfected/transduced cell lines. NADPH formation was monitored by absorbance measurements at 340 nm. Note that IDH1^R314C-BAPHIS was far less effective in oxidative carboxylation of isocitrate than IDH1^WT-BAPHIS. (C) Km values of IDH1-GST for NADP⁺. The Km value for NADP⁺ for IDH1^R132H-GST could not be determined. (D) D-2-HG production by IDH1-BAPHIS expressing cell lines was measured with LC-MS. Only IDH1^R132H-BAPHIS expressing cells were capable of D-2-HG production.