Pvdf filter

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.

Proteins were extracted by homogenizing animals in RIPA buffer supplemented with 10% protease inhibitor cocktail (Sigma-Aldrich). The homogenates were cleared by centrifugation, and the supernatants were subjected to electrophoresis through 10% SDS-polyacrylamide (SDS/PAGE) gels containing pre-stained protein markers and transferred to PVDF filters (Bio-Rad Laboratories, Hercules, CA (Harlow and Lane 1988 ). The filters were incubated with 5% non-fat dry milk in TBST buffer (50 mM Tris-HCl, ph 7.6, 100 mM NaCl, 0.1% Tween-20) to block non-specific binding, washed three times for 5 min in TBST, and incubated overnight in a 1:100 dilution of PIWI polyclonal antibody (ab5207, Abcam, Cambridge, MA) in TBST at 4°C, and finally incubated for 1 hr at room temperature in a 1:25000 dilution of HRP-conjugated anti-rabbit IgG secondary antibody. After three washes for 5min with TBST, the signals were visualized with Chemiluminescence Luminol (Santa Cruz Biotechnology, Santa Cruz, CA) used according to the supplier’s specifications. Images of X-ray films were taken with a digital camera, scanned, and band density was calculated by comparison with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gel loading standard (Sigma-Aldrich).