Western Blot Analysis of Macrophage Proteins

Macrophages were harvested, and proteins were extracted using RIPA buffer (Beyotime) containing protease inhibitors PMSF (Servicebio, China) and cocktail (Roche). For the protein preparation of the culture supernatants, 700 μL culture supernatants were collected and mixed with 700 μL 100% methanol and 175 μL trichloromethane. Supernatants were centrifuged at 13,000 rpm for 5 min at 4 °C to precipitate the proteins. The remaining proteins were resuspended in 20 μL10% SDS. 30 µg of cell lysate samples or culture supernatant samples were resolved by a 12% SDS/PAGE. Western blotting was performed as described previously [34 (link)]. The blots were visualized by ChemiDoc XRS (Bio-Rad, USA). The relative band intensity was quantified using the Image Lab Analyzer software (Bio-Rad). α-tubulin was used as the loading control. The antibodies used in the study are shown in Table 1.