Western Blot Analysis of TAP1 Protein

Proteins were extracted from 769P and 786O cells using Western and IP lysis buffer (Beyotime Biotechnology, Jiang Su, China), as previously mentioned (40 (link)). Samples were separated by electrophoresis on 10% SDS gel (epizyme, Shanghai, China) and then transferred to a methanol-activated polyvinylidene fluoride (PVDF) membrane (Millipore, Shanghai, China). Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and then incubated with primary antibodies, anti-TAP1 (1:1,000, 11114-1-AP, proteintech, China), and anti-beta-Actin primary antibody (1:1,000, AC026, Abclonal, China) at 4°C for 8 hours. After washed with TBST for three times, membranes were incubated with secondary antibody goat anti-rabbit IgG conjugated with HRP (1:3,000, SA00001-2, Proteintech, China) at room temperature for 60 min. After three washes with TBST for each 10 mins, the bands were visualized using ECL-plus™ western blotting chemiluminescence kits (BD Biosciences, NJ, USA). Image J was used to analyze strip grayscale, and Actin was used as a reference.