Fig 1: The effect of adding human TrpRS protein to the assay buffer on [3H]Trp uptake into HeLa cells overexpressing 6×His-tagged human indoleamine 2,3-dioxygenase 1 (IDO1). Control vector, human wild-type (WT) IDO1 or H346A IDO1 mutant expression vector was transfected into cells. (A) Western blot analysis of extracts from 6×His-tagged human IDO1-overexpressing cells using anti-IDO1, anti-6×His tag, and anti-ß-actin antibodies. The arrows indicate the expected position of IDO1 and ß-actin. Molecular size markers (kDa) are shown on the left. (B) [3H]Trp uptake into HeLa cells, in which human WT IDO1 or H346A IDO1 mutant was overexpressed, in the absence or presence of human TrpRS protein. All data are expressed as mean ± SD of at least four independent experiments. ** p < 0.01, * p < 0.05. (C) Western blot analysis of extracts from 6×His-tagged human IDO1-overexpressing cells using anti-activating transcription factor 4 (ATF4) or anti-ß-actin antibodies. The arrows indicate the expected positions of ATF4 and ß-actin. Molecular size markers (kDa) are shown on the left. (D) Western blot analyses of extracts of 6×His-tagged human WT IDO1-overexpressing HeLa cells, which were incubated in normal medium with additional Trp (0 or 400µM Trp) for 18 h, by using anti-ATF4, anti-IDO1, and anti-ß-actin antibodies. Final concentration of Trp in the medium is 80 or 480 µM, because normal medium contains 80 µM Trp.