Enhanced chemiluminescence system

Total cell extracts were lysed on ice using a lysis buffer (containing proteinase and phosphatase inhibitors; Beyotime Biotech Co., Ltd.). Protein concentrations were measured using the BCA Kit (Vazyme Biotech Co., Ltd.). Samples were mixed with SDS-PAGE Sample Loading Buffer (5X, Beyotime Biotech Co., Ltd.) and denatured at 95°C for 5 min, then resolved on 10% gels using SDS-PAGE in running buffer (Sangon Biotech Co., Ltd. Tris, 3 g; Glycine, 14.4 g; SDS, 1 g; Fix the volume to 1 liter with DD water.). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% skimmed milk at room temperature for 1 h, the PVDF membranes were incubated with primary antibodies at 4°C overnight, washed with PBST and then incubated with secondary antibodies at room temperature for 1 h. After washing with PBST 3 times, signals were detected using an enhanced chemiluminescence system (Vazyme Biotech Co., Ltd.) and acquired by ChemiScope 3300 Mini Imaging System (Clinx Science Instruments Co., Ltd.).

The total protein was extracted and subjected to electrophoresis. Cell lysates were prepared by using RIPA protein extraction reagent (Beyotime Biotechnology) and protein concentrations were determined by Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Samples containing equal amounts of protein were separated by 10% SDS‐PAGE and transferred onto PVDF membranes (Amersham Hybond). Membranes were blocked by 5% BSA in TBS/0.1% Tween‐20, then incubated overnight at 4°C. The expression level of each protein was detected by an enhanced chemiluminescence system (Vazyme Biotech). The primary Abs anti‐PD‐L1, anti‐UCHL1, anti‐STAT1, anti‐phospho‐STAT1, anti‐ERK1/2, anti‐phospho‐ERK1/2, anti‐P65, and anti‐phospho‐P65 were purchased from Cell Signaling Technology with catalog numbers E1L3N, D3T2E, 7649P, 9175S,4695P, 4370S, 3033T, and 8242T, respectively. The primary Abs anti‐AKT and anti‐phospho‐AKT were purchased from Epitomics with catalog numbers 1085‐S and 2214‐S, respectively. The primary Abs also included anti‐Flag (Cat# F1804; Sigma‐Aldrich) and anti‐β‐actin (Cat# PR‐0255; ZSGB‐BIO). The secondary Abs were goat anti‐rabbit IgG Ab or goat anti‐mouse IgG Ab (Cat# ZB‐2301 or Cat# ZB‐2305; ZSGB‐Bio).