Cd206 mrc1

After lysing the cells in Radio-Immunoprecipitation Assay (RIPA) lysis buffer (Pierce, Rockford, IL, USA) supplemented with protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich Corporation, St. Louis, MO, USA), 20μg protein lysate samples were loaded per lane into 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad); after electrophoresis, the separated protein blots were transferred onto nitrocellulose membranes (Millipore Corporation, Bedford, MA, USA), membranes were incubated with 5% skimmed-milk in Tris-buffered saline with Tween 20 (TBST) for 1 h to block non-specific binding, and then incubated overnight at 4 °C with primary antibody against CD80 (1:1000, #15416, Cell Signaling Technology, Inc., Danvers, MA, USA), CD206/MRC1 (1:1000, #91992, Cell Signaling Technology, Inc., Danvers, MA, USA), or β-actin (1:1000, #3700, Sigma-Aldrich Corporation, St. Louis, MO, USA), washed in TBST three times and then incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5000) or anti-mouse (1:3000) secondary antibody (Millipore Corporation, Bedford, MA, USA). Thereafter, Pierce enhanced chemiluminescence (ECL) western blotting substrate (Pierce) was used for band detection and imaging done with the BioSpectrum Imaging System (UVP, Upland, CA, USA).

ELISA, immunoblot, and mRNA analysis were conducted as described [62 (link), 63 (link)]. ELISA kits included IFN-γ (Invitrogen, 88–8314-22), IL-1β (eBioscience, 88,701,388), IL-18 (MBL, D042-3), IL-6 (BioLegend, 431,301), and TNF-α (BioLegend, 430,901). Primary antibodies used in this study include antibodies against ALIX (Cell Signaling, 2171, 1:1000), TSG101 (Genetex, GTX70255; 1:500), GAPDH (Cell Signaling, 2118; 1:1000), CD206/MRC1 (Cell Signaling, 24,595; 1:1000), occludin (ProteinTech, 27,260-1-AP; 1:1000), claudin1 (ProteinTech, 13,050-1-AP; 1:1000), CD81 (ProteinTech, 27,855-1-AP; 1:1000), Cxcl10 (ProteinTech, 10,937-1-AP; 1:1000), Arg1 (ProteinTech, 66,129-1; 1:1000), FMOD (ProteinTech, 60,108-1; 1:500), and MFGE8 (ThermoFisher, PA5-109,955; 1:1000).