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. Author manuscript; available in PMC: 2018 Aug 10.
Published in final edited form as: Biochem J. 2017 Aug 10;474(16):2829–2839. doi: 10.1042/BCJ20170389

Figure 2. IDH2 Acetylation and Canonical vs. 2-HG Generating Activity.

Figure 2

(A): Purified IDH2 was acetylated in-vitro with acetyl-CoA (see methods), followed by western blot probe with anti-acetyl-lysine (K-Ac) antibody. Ponceau S stained membrane below indicates protein loading. Images representative of at least 4 independent experiments. (B): Canonical activity of naïve and acetylated IDH2 (Ac-IDH2) with isocitrate and NADP+ as substrates was measured spectrophotometrically as NADPH production (left panel) or by LC-MS/MS as α-KG production (right panel). (C): Non-canonical reductive activity of IDH2 with α-KG and NADPH as substrates was measured spectrophotometrically as NADPH consumption (left panel) or by LC-MS/MS as 2-HG production (right panel). All enzyme rate data are means ± SEM, n=4. *p<0.05 between naïve and Ac-IDH2. Reactions monitored are shown above each graph, with the metabolite measured shown in red font.