Quantification of IDO1 Expression in Breast Cancer Cells

This procedure was carried out as described previously [69 (link),71 (link),72 (link)]. Breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in a SDS lysis buffer (60 mM Tris-HCl (pH 6.8 at 25 °C), 2% (w/v) SDS, 10% glycerol). Protein concentration was quantified using the bicinchoninic acid reagent (Sigma). Proteins (25 μg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to PVDF membranes (Millipore Merck, Auckland, New Zealand). Membranes were immunoblotted with antibodies against human IDO1 and tubulin (Sigma). Bound antibody was visualized using Pierce™ ECL Western Blotting Substrate (ThermoFisher Scientific) and the chemiluminescence measured using image analyser LAS-3000 (Fujifilm, Tokyo, Japan).