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. 2016 Sep 6;113(38):E5598–E5607. doi: 10.1073/pnas.1609221113

Fig. 2.

Fig. 2.

Mutations in GPT2 lead to reduced protein levels and enzyme activity. (A) Western blot results of mutated GPT2 protein. Lysates of HeLa cells transfected with human control GPT2 (hGPT2-V5His), p.Arg404*, p.Ser153Arg, p.Pro272Leu, or vector-only constructs were probed with anti-GPT2 antibody. All of the three GPT2 mutants show reduced protein levels compared with control GPT2 (hGPT2-V5His). Arrow indicates the expected GPT2 protein band; arrowhead indicates the truncated GPT2 protein band. (B) Quantification of Western blots. (Left) Overexpressed GPT2 bands were normalized to tubulin. (Right) Overexpressed GPT2 level in each lane was normalized to the GPT2 control (hGPT2-V5His). n = 6 replicates. Data are presented as means ± SEMs. ***P < 0.001. (C) GPT enzyme functional assay indicated that mutations in GPT2 lead to loss of the enzyme activity. HeLa cells were transfected with human control GPT2 (hGPT2-V5His), p.Arg404*, p.Ser153Arg, p.Pro272Leu, or vector-only constructs. Lysates were used to analyze the GPT activity. (Left) GPT activity was measured by pyruvate production after either a 20- or 40-min incubation with substrate at 37 °C. All three GPT2 mutants had similar pyruvate production as the vector control. (Right) GPT activity was measured in cell lysate after incubating with substrate at 37 °C for 40 min and is represented as enzyme activity (in milliunits) per milligram of tested sample. Only the overexpressed GPT2 control protein showed significant high enzyme activity. n = 4 replicates. Data are presented as means ± SEMs. ***P < 0.001.