Polyvinylidene difluoride membranes

Mouse tumor tissues were lysed using a lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and quantified using a bicinchoninic acid protein assay. Aliquots containing 20 µg protein were dissolved in Laemmli buffer and incubated at 95°C for 10 min. Proteins were separated on a 12% Tris-glycine SDS-PAGE (Beyotime Institute of Biotechnology), transferred onto polyvinylidene difluoride membranes (BD Pharmingen, San Diego, CA, USA) and incubated with 5% non-fat dry milk in Tris-buffered saline/0.2% Tween-20 (TBST) for 2 h at room temperature. The membranes were then incubated with anti-B7-H4 antibody (1:1,000 dilution; cat. no. ab108336; Abcam, Cambridge, UK) at 4°C overnight. Membranes were then washed three times with 1X TBST followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:5,000; dilution; cat. no. ZB-2301; BIOSS, Beijing, China) for 1 h at room temperature. Membranes were washed five times in 1X TBST and proteins were visualized using an enhanced chemiluminescence kit (Roche Diagnostics, Basel, Switzerland). GAPDH was detected with mouse anti-GAPDH antibody (1:1,000 dilution; cat. no. AB-P-R 001; OriGene Technologies, Inc., Beijing, China) as an internal control.

Total protein was collected with Total Protein Extraction Kit (KeyGen Biotech. Co. Ltd., Nanjing, China). Proteins (30 mg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel, and transferred electrophoretically onto polyvinylidene difluoride membranes (BD Biosciences). The membranes were blocked in 5% skimmed milk for 1 h, and then incubated for 2 h with rabbit anti-STAT6 (1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-phosphorylated (p)-STAT6 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) or rabbit anti-GAPDH (1:1,000; Bioworld, Technology, Inc., St. Louis Park, MN, USA), which was used as loading control. Following incubation with goat anti-rabbit secondary antibody (Bioworld, Technology, Inc.), the signals derived from the antibody-antigen binding reaction were detected by enhanced chemiluminescence (BD Biosciences).