Ab79483

GC tissues or cells were collected, and the total protein was extracted by radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration was determined using a bicinchoninic acid (BCA) kit (20201ES76, Yeasen Biotechnology, Shanghai, China). Quantification was performed according to different concentrations. Following separation with the use of polyacrylamide gel electrophoresis, the protein was transferred onto a polyvinylidene fluoride membrane (IPVH85R, Millipore, Darmstadt, Germany), which was then blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. Next, the membrane was incubated with primary rabbit antibodies (Abcam, Cambridge, UK) to CXXC4 (ab105400, 1:100), ERK (ab184699, 1:10,000), phosphorylated ERK (ab79483, 1:1,000), PD-L1 (ab233482, 1:100), and GAPDH (ab128915, 1:10,000) overnight at 4°C. The following day, the membrane was cultured with horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) (ab205718, 1:20,000, Abcam, Cambridge, UK) for 1 h at room temperature. The membrane was visualized using the developing solution. Protein quantitative analysis was conducted using ImageJ 1.48u software (National Institutes of Health) and expressed as the gray value ratio of each protein to the internal reference GAPDH.

Primary cortical neurons or brain tissues were lysed with RIPA buffer containing phosphatase and protease inhibitors. After measurement with BCA kits, the proteins were denatured by boiling for 10 min and then loaded on 8-12% SDS‒PAGE gels, followed by transfer from the gels to activated PVDF membranes. After blocking with 5% nonfat dry milk, the membranes were incubated with the following primary antibodies: anti-PI3K (ab154598, 1:2000), anti-p-Akt (ab38449, 1:1000), anti-Akt (ab283852, 1:2500), anti-GRP78 (ab21685, 1:3000), anti-p-PERK (ab192591, 1:1000), anti-PERK (ab79483, 1:2500), and anti-p-IRE1α (ab124945, 1:2500) were obtained from Abcam, USA and anti-IRE1α (#3294, 1:2500), anti-ATF6 (#65880, 1:2000), anti-CHOP (#5554, 1:2000), anti-Caspase12 (#35965, 1:2500), anti-Caspase3 (#9579, 1:1000), anti-Bcl-2 (#3869S, 1:2000) and anti-Bax (#5023, 1:2500) were supplied by Cell Signaling Technology, U.S.A. Anti-β-actin antibody (GB15004, 1:3000) was purchased from Servicebio Technology (China). After incubation with the primary antibodies overnight, the membranes were washed three times with TBST and incubated with secondary anti-rabbit HRP antibody (GB1213, 1:5000, Servicebio Technology, China). Enhanced chemiluminescence was used to visualize the membranes, and the analysis was performed using ImageJ software.