Ifnar1 antibody

Flow cytometry was performed as previously described.^{24 (link)} in brief, HN4 and HN30 cells were incubated with the indicated agent for 48 h. The cells were collected and incubated with anti-human PDL1 antibody at 1:100 (BD Biosciences, Franklin Lakes, NJ) for 30 min on ice. Then, the cells were resuspended in 100 μl fluorescence-activated cell sorting buffer and analysed on BD Fortessa flow cytometer. The final results were analysed with FlowJo software. Signal intensity was calculated as the ratio of the median fluorescence of the PDL1 antibody to that of the isotype control antibody (SFI: specific fluorescence index). CD4-FITC antibody, CD8-PerCP-Cy5.5 antibody, CD56-APC antibody, and PD1-PE antibody (all purchased from BD Biosciences) were applied to detect the PD1 expression on the surface of immune cells from peripheral blood of HNSCC patients and healthy controls. The IFNAR1 antibody (Abcam, Cambridge, MA, UK) and PE-conjugated secondary antibody (Proteintech, Rosemont, IL, USA) were used to analyse the surface IFNAR1 expression on immune cells.

Cells were lysed in M-PER Mammalian Protein Extraction Reagent (Thermo scientific) containing the protease inhibitor cocktail, 1mM PMSF, 1mM sodium orthovanadate, and 1mM sodium fluoride for 10 minutes on ice followed by a brief sonication. Cell lysates were cleared by centrifugation at 14,000 x g for 2 minutes. The supernatant was collected, boiled in Laemmli buffer for 5 minutes, and used for immunoblot analysis or stored at −80°C for future use. The rabbit anti-HCV core antibody was prepared in our laboratory [34 (link)]. TNFR1, p65 NF-κB, PARP, Caspase-8, and IRAK1 antibodies were from Cell signaling, and actin and IFNAR2 antibodies were from Sigma. IFNAR1 antibody was from Abcam, and GAPDH, TLR7, TLR8, and TRAF6 antibodies were from Santa Cruz. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and rabbit anti-mouse secondary antibodies were also purchased from Abcam.