Identification of IDO transfected CHO cells. (A) Identification of recombinant plasmid pIRES2-EGFP-IDO by restriction enzyme analysis. The plasmid pIRES2-EGFP-IDO can be digested with Bgl IIand Sal I. xperiments in this figure and following figures were performed at least three times on separate occasions. (B) IDO gene integration and transcription by PCR and RT-PCR. (C) Western blot analysis of IDO protein expression in CHO-IDO cells using anti-IDO antibody. In transfected group, CHO cells transfected with IDO expressed the 42 kDa IDO protein, indicating that CHO cells stably transfected with IDO could produce IDO protein. (D) Analysis of free amino acids in culture supernatant. Amino acid level in CHO cells 72 h after IDO transfection: (His) 33.75 mg/L, (Kyn) 7.03 mg/L, (Trp) < 3 pmol. Amino acid level in CHO cells with p IRES2-EGFP transfection 72 h after culturing: (His) 38.12 mg/L, (Trp) 5.63 mg/L, (Kyn) < 3 pmol. His: histidine; Trp: trytophan; Kyn: kynurenine.