Western blot analysis was performed on whole cell extracts (30 µg lysate) of HEK-293 (Lane 1), HEK-293 treated with hIFN-γ (50 ng/mL, for 24hrs) (Lane 2), MDA-MB-231 (Lane 3), MDA-MB-231 treated with hIFN-γ (50 ng/mL, for 24hrs) (Lane 4), HT-1080 (Lane 5) and HT-1080 treated with hIFN-γ (50 ng/mL, for 24hrs) (Lane 6). The blots were probed with Anti-IDO1 Recombinant Rabbit Polyclonal Antibody (Product # 711778, 1 µg/mL). A 43 kDa band corresponding to IDO1, that specifically increases upon hIFN-γ treatment is as shown. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).