PDAC Tissue Western Blotting Analysis

Western blotting was conducted as previously reported 22 (link). Briefly, we consistently collected PDAC surgical resection pathological specimens without any bias or pre-screening during 2019, from which 42 paired PDAC tissues and paracancer tissues were randomly selected for western blot analysis. 100 mg samples were lysed in radioimmunoprecipitation assay (RIPA) buffer (APPLYGEN, C1053+) containing 1% protease inhibitor and 1% phosphatase inhibitors for 30min on ice. Then, centrifugation at 12,000 × g for 15 min at 4 °C, the supernatant proteins were quantified using BCA assay (Thermo Scientific, BCA protein assay kit, 23225). Thirty micrograms of protein were mixed with 5 × loading buffer and denatured in a 95 °C water bath. Primary antibodies against TIGIT rabbit mAb (Abcam, ab243903, 1:1000), CD155 rabbit mAb (Cell Signaling Technology, #81254, 1:1000), and anti-β-Actin (D6A8) rabbit mAb (Cell Signaling Technology, #8457, 1:1000) were incubated with PDAC tissues overnight at 4°C for the following blot detection.