High efficiency ripa lysis buffer

High-efficiency RIPA lysis buffer supplemented with 1% protease inhibitor and 1% phosphorylase inhibitor (Beyotime, Shanghai, China) was adopted for total protein extraction from tissues and cells. Following protein concentration measurement using a BCA kit (Thermo Fisher Scientific), sample proteins were separated by 10% SDS-PAGE, transferred to a PVDF membrane, and then blocked with 5% BSA. Subsequently, the protein-loaded membrane was probed overnight at 4 °C with primary antibodies to UCHL3 (rabbit, 1:2000, ab241490, Abcam), AhR (mouse, 1:500, MA1-513, Thermo Fisher Scientific), PD-L1 (rabbit, 1:1000, ab205921, Abcam) and β-actin (rabbit, 1:5000, ab8227, Abcam; loading control), followed by 1.5-h incubation with HRP-labeled goat anti-rabbit IgG (1:20000, ab205718, Abcam) or HRP-labeled goat anti-mouse IgG (1:500, 31430, Thermo Fisher Scientific) at room temperature. The blots were visualized using developing solution (NCI4106, Pierce, Rockford, IL). Quantitative analysis was then conducted using ImageJ 1.48u software (Bio-Rad, HercμLes, CA).

Mouse liver and ileum tissues were lysed in high-efficiency RIPA lysis buffer (Beyotime, China) on ice. The tissue lysates were centrifuged at 12,000 rpm at 4°C for 15 min to obtain the protein-containing supernatant. Protein concentration was determined using the BCA protein quantification assay kit (Applygen, China), and 50 µg of protein was loaded per well for SDS-PAGE gel electrophoresis. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel electrophoresis was performed, and the proteins were then transferred to an activated polyvinylidene difluoride (PVDF) membrane using transfer buffer containing distilled water and methanol. The PVDF membrane was subsequently blocked with 5% skim milk and incubated with the respective primary antibodies overnight at 4°C, with β-actin serving as the internal reference protein. The membrane was then incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) (Goat anti-Rabbit IgG, HRP Conjugated, Cwbio, 1:5000) at room temperature for 1 h. Enhanced chemiluminescence (ECL) plus (Beyotime, China) was applied to the membrane surface, and the membrane was placed in the Molecular Imager Gel Doc XR + System (Bio-Rad, United States) for image acquisition. Image Lab software was used to acquire images, and subsequently, ImageJ was employed for image analysis.