Macrophage and Fibroblast Signaling Pathways

The measurement of signaling pathways involved in macrophage polarization and fibroblast activation was achieved by WB. Cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Haimen, China) supplemented with protease and phosphatase inhibitor cocktail (Pierce, Thermo Fisher Scientific) for 30min on ice, and then centrifuged at 15,000×g for 30 min at 4°C. Cell extracts were heated for 5 min at 100°C. The proteins were analyzed by Western blot as previously described.17 (link) p-Smad3 (1:1000; Affinity, AF3362), Smad3 (1:1000; Affinity, AF6362), GAPDH antibody (1:1000; Affinity, AF7021), and Goat anti-rabbit IgG (1:5000; Affinity, S0001) were used according to the manufacturer’s protocol. Immunoreactive bands were detected with electrochemiluminescence (ECL) reagent (Beyotime Biotechnology) and imaged with a Tanon-5200 Multi Fluorescence imager.