Turbo trans system

For the assessment of PD-L1 expression (n=4-7), crude membranes were extracted using Mem-PER™ Plus Membrane Protein Extraction Kit (ThermoFisher Scientific) according to the manufacturer's instructions. Equal amounts of protein (10 μg) were resolved in 4-20% Mini-PROTEAN polyacrylamide gel (BioRad, CA, USA) and were subsequently transferred onto a nitrocellulose membrane using a BioRad Turbo Trans system. After blocking (5% non-fat dry milk), membranes were incubated at 4°C overnight with anti-mouse PD-L1 (1:1000, Sino Biological, 50010-732). anti-mouse GAPDH (1:10000, Invitrogen, AM4300) and anti-Na+/K+ ATPase (1:3000, Abcam, ab7671 and 1:1000, Cell Signalling Technology, 3010). This was followed by incubation with secondary antibodies conjugated to horseradish peroxidase (1:10000, rabbit or goat anti-mouse, Jackson ImmunoResearch Inc., PA, USA) at room temperature for 1h. The blots were developed using the ECL kit (Thermo Scientific, Rockford, IL) and imaged by Amersham imager 600 system (GE Healthcare Bio-Sciences, Uppsala, Sweden). Na+/K+ ATPase was used as a loading control. Densitometric analyses were performed using ImageJ 1.51 software (NIH), and the data were normalized relative to appropriate GADPH controls.

Macrophages were lysed in lysis buffer (T-PER, Thermo Fisher Scientific) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) for 30 min on ice. Cell debris was removed by centrifugation at 15,000×g for 10 min at 4 °C. Protein concentration in the cell lysate was determined using a BioRad protein assay kit (BioRad, Hercules, California, USA). The proteins in each sample (10 µg) were separated by 10% SDS-PAGE, and subsequently transferred onto a nitrocellulose membrane using the BioRad Turbo Trans system. After blocking with 5% skim milk for 1 h in TTBS (20 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.5), membranes were incubated with primary antibodies, anti-phospho STAT1-Y701 (1:1,000 dilution, Cell Signaling, Beverly, MA, USA), or mouse anti-human β-actin (1:3,000 dilution, Thermo Fisher Scientific) overnight and then for 1 h with the respective secondary antibodies (1:3,000 dilution, goat anti-rabbit and goat anti-mouse HRP conjugated, Jackson Immuno Research, USA). Blots were developed using Super Signal West Pico (Thermo Fisher Scientific), and signals were detected with Amersham Imager 600. Intensity of the bands was quantified by ImageJ densitometry with β-actin as a loading control.