Chemidoc touch gel imaging system

About 1 × 10⁶ cells were lysed with 100 μL RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA, # 89900) at 4 °C for 30 min. The lysed cells were spun at 14,000 rpm at 4 °C for 10 min, and the supernatant was saved.
About 20 μg of cell lysate was loaded on a 7.5% SDS-PAGE and was then electroblotted onto PVDF membrane (Millipore, Burlington, MA, USA, #IPVH00010). The membrane was firstly blocked with 5% nonfat dry milk in TBST buffer (0.05% Tween-20; 10 mM Tris-buffer, pH 7.5; and 150 mM NaCl) for 1 h at room temperature and was then incubated with 1:1000 primary antibody mouse anti-P-gp (Santa Cruz, Dallas, TX, USA, #SC-55510,) or 1:3000 mouse anti-β-actin (Santa Cruz, #SC-47778) at room temperature for 1 h. Subsequently, the membrane was incubated with 1:3000 secondary antibody goat antimouse-IgG-HRP (Santa Cruz, #SC-516102) for 1 h at room temperature. A chemiluminescent substrate (Millipore, WBKLS0500) was added to the membrane, and its signal was detected using ChemiDoc^TM Touch Gel Imaging System (Bio-Rad, Hercules, CA, USA, #1708370). In the Western blot membrane, total P-gp and β-actin expression in each lane was quantified with ImageJ software. Relative P-gp expression was an intensity ratio of P-gp band relative to the β-actin band.

Control or treated groups were dry pelleted after 48 h of treatment and lysed with equal volumes of 1× lysis buffer (1 mM PMSF and 1 X protease Inhibitor Cocktail) on ice for 30 min. Then, they were sonicated and centrifuged at 18000 G for 20 min at 4 °C. Protein concentrations were determined by Bradford protein assay. Equal amounts of proteins (30 µg) were separated by 12% SDS PAGE gel electrophoresis and then transferred to PVDF membranes that were blocked in PBS 5% BSA containing 0.1% Tween 20 at room temperature for 1 h. Afterward, the membranes were incubated with primary antibodies against PD-L1 (1:200) (Santa Cruz: Biotechnology) or α-tubulin (1:5000) (Cell Signaling) overnight at 4 °C. The next day, membranes were washed (PBS-0.1% Tween) and incubated with HRP-secondary antibody (1:5000) at room temperature for 1 h. After washing, the protein bands were detected with a chemiluminescence detection system (ChemiDoc^TM Touch Gel Imaging System—Bio-Rad Laboratories), which were quantified and numerated using Fiji software (Rasband, W.S., ImageJ). A ratio was calculated for PD-L1 expression/α-tubulin expression, and then a second ratio was calculated for test/control to compare expression of treated to untreated cells.

Western and IP cell lysates (Beyotime, Shanghai, China) was used to obtain the total protein. The protein extracted was mixed 1:1 with 2× loading buffer solution, which was denatured. The loading of sample to be tested was 10 µL and the Multicolor Prestained Protein maker (Epizyme, Shanghai, China) was 5 µL in every gel lane for electrophoretic analysis. The gels in Coomassie brilliant blue (CBB) solution were stained, and further used for Western blotting analysis. The Rubisco large subunit with CBB staining was used as protein loading controls. The protein transferred to PVDF membrane was performed by wet transfer method. The anti-GFP rabbit polyclonal antibody and HRP-labeled Goat anti-Rabbit IgG (Sangon Biotech, Shanghai, China) were used for detection at a 1:5000 dilution, respectively. Western Bright ECL HRP (Advansta, San Jose, CA, USA) color solution was added to the surface of the PVDF membrane, which placed at room temperature and incubated away from light for 3 min. The ChemiDocTM Touch gel imaging system (Bio-Rad, Hercules, CA, USA) was used to take pictures.