Volume one software

Membrane proteins were isolated with Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific), and cytoplasmic proteins were extracted with NE-PER™ Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific). We quantified the proteins with the DC Protein Assay kit (Bio-Rad, Hercules CA, USA). Denatured proteins were separated on a 10% SDS-PAGE gel and then transferred to PVDF filters (Bio-Rad). After blocking with 5% non-fat milk-TBST (Bio-Rad), the blots were incubated mouse PD-L1 antibody (Abcam), human PD-L1 antibody (Cell signaling), β-actin antibody (Cell signaling), CD63 polyclonal antibody (Abcam), or Calnexin polyclonal antibody (Abcam) at 4°C overnight, respectively. After washing 3 times with 1×TBST (Bio-Rad), the blots were incubated with peroxidase-linked secondary goat anti-rabbit IgG antibody (Bio-Rad), or goat-anti-mouse IgG antibody (Bio-Rad) were incubated for 1 h at room temperature. Finally, after incubation with SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific, Worcester, MA, USA), visual imaging was performed by VersaDoc™ Imaging System (Bio-Rad). Volume One software (Bio-Rad) was used for the relative quantification of blotted proteins.