Western Blotting

Total protein was extracted from RAW 264.7 cells using the same procedures as described in detail elsewhere (Santa et al., 2016). Briefly, raw cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo, 89901) containing proteinase inhibitors and phosphatase inhibitors (KeyGEN BioTECH, KGP250). The protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, 23,225). Subsequently, the phenol–ethanol supernatant was mixed with isopropanol to isolate proteins. Equal amounts of protein and supernatants were separated by 12.5 or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.45 or 0.20 μm-pore polyvinylidene fluoride membranes (Merck Millipore, IPVH00010 or ISEQ00005). Membranes were incubated overnight with antibodies against CD163 (Abcam, ab182422), CD206 (Abcam, ab64693), PDGF-BB (Santa Cruz, sc365805), MMP9 (Abcam, ab38898), and β-actin (Cell Signaling echnology, 2118). The membranes were then incubated with peroxidase-conjugated goat antirabbit immunoglobulin G (IgG) (h + l) secondary antibody (1:5,000) for 1 h. Protein signals were detected using an enhanced chemiluminescence kit (Cat. WBKLS0500, Millipore), and Western blot bands were examined and analyzed with a chemiluminescence instrument (Guangzhou Ewell Bio Technology Co. Ltd., China).