Ab241490

Paraffin-embedded Sections (4 μm thick) of tissues were subjected to antigen retrieval in EDTA buffer, followed by incubation with 0.3% H₂O₂. After blocking with 5% BSA, samples were immunoblotted at 4 °C overnight with primary antibodies to UCHL3 (1:100, ab241490, Abcam, Cambridge, UK), AhR (1: 200, MA1-513, Thermo Fisher Scientific) and ki67 (1:100, ab15580, Abcam). Subsequently, the sections were incubated with the biotinylated secondary antibody goat anti-rabbit IgG (1:1000, ab6721, Abcam) or goat anti-mouse IgG (1: 500, 31430, Thermo Fisher Scientific), followed by another incubation with HRP-labeled streptavidin (Innova Biosciences, Cambridge, UK). After DAB development and hematoxylin counterstaining, the sections were observed using a microscope (Leica-DM2500, Leica, Bensheim, Germany), and images were photographed by ImagePro Plus 7.1 software.

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).

Co-IP assay was performed to evaluate the interaction between UCHL3 and AhR. HEK293T cells (ATCC) were lysed in IP lysis buffer (P0013). Next, the cell lysate containing 200 μg of protein was then incubated with Dynabeads^® protein G magnetic beads at 4 °C for 4 h, with 2 μg of anti-AhR antibody (sc-133088, Santa Cruz Biotechnology, Santa Cruz, CA) or IgG (as the NC) added in. Then, the precipitated protein complexes underwent western blot analysis with the anti-rabbit antibody against UCHL3 (1:2000, ab241490, Abcam).