Ab105400

The treated cells were collected and washed twice with PBS buffer. Cells were then lysed by RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) for total protein extraction. The BCA protein assay kit (Beyotime Biotechnology) was applied for determining protein concentration. Protein with equivalent amounts were subsequently separated by SDS/PAGE and then transferred on to a PVDF membrane. After blocking, the membranes were incubated with primary antibodies against CXXC4 (ab105400, 1:500), BCL-2 (ab32124, 1:1000), Bax (ab32503, 1:1000), and β-actin (ab8227, 1:3000) (Abcam, Cambridge, U.K.) overnight at 4°C. After that, the membranes were incubated with secondary antibody (ab205718) (Abcam) and exposed using an Ultrasensitive ECL Chemiluminescence kit (Sangon Biotech) according to the manual.

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.