Mfge8

Human PASMCs were cultured and stimulated, fixed in 4% paraformaldehyde, permeabilized with phosphate buffered saline (PBS) containing 0.1% Triton‐X and blocked with blocking buffer. Cells were incubated with the primary antibody against MFG‐E8 (Abcam) and FITC‐conjugated anti‐mouse IgG (Jackson ImmunoResearch, West Grove, PA), with subsequent washing to remove the nonspecific binding. Cell nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). Positively stained cells were observed by confocal laser scanning Immunofluorescence microscopy (Nikon, Tokyo, Japan).

For protein preparation, cultured cells and mice skin specimens (using a manual pestle homogeniser) were lysed in an appropriate volume of cold RIPA buffer plus protease inhibitors. BCA Kit (Beyotime, China) was used to measure protein concentration. Extracted protein (20 μg) was separated by SDS-polyacrylamide electrophoresis (SDS-PAGE) before being transferred onto polyvinylidene fluoride (PVDF) membranes. For immunodetection, primary antibodies specific for CD63(Abcam, UK), TSG101(Abcam, UK), MFGE8(Abcam, UK), Bax (Abcam, UK), Bcl-2(Abcam, UK), CD206(Abcam, UK), Arg-1 (Abcam, UK), iNOS (Abcam, UK), TGFB1 (Abcam, UK), SMAD3 (Abcam, UK) and GAPDH (Abcam, UK). IgG secondary antibody labelled by horseradish peroxidase (Abcam, UK), and enhanced chemiluminescence were used.