Mouse anti human β actin mab

LLC cells were lysed using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing PMSF protease inhibitor (1 mmol/l). Protein concentration was determined by using the bicinchoninic acid protein assay (Bio-Rad Laboratories, Inc.). Proteins (30 µg) were separated by 8% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk and 3% BSA in TBST (0.25% Tween-20) and subsequently incubated overnight at 4°C with the following primary antibodies: Mouse anti-human IDO1 mAb (cat. no. sc-53978; 1:200; Santa Cruz Biotechnology, Inc.) and mouse anti-human β-actin mAb (cat. no. sc-47778; 1:2,000; Santa Cruz Biotechnology, Inc.). The membranes were washed three times with TBST and incubated with the secondary antibody, goat anti-mouse IgG-HRP (cat. no. sc-358914; 1:5,000; Santa Cruz Biotechnology, Inc.), at room temperature for 2 h. Enhanced chemiluminescence reagent (OriGene Technologies, Inc.) was used to detect the signal on the membrane. The relative expression levels of the IDO1 protein were calculated using the gray scale ratio of IDO1/β-actin using ImageJ version 1.46 software (National Institutes of Health).