Western Blotting of Microglial Proteins

Western blot analysis was carried out as described previously [20 (link)]. Total protein was extracted from BV2 microglia. Equal amounts of protein were separated on sodium dodecyl sulfate polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and blocked with 5% bull serum albumin for 1 h at 25 °C. Membranes were incubated overnight with an antibody against iNOS (1:800, Abcam, Inc., Boston, MA, USA), an antibody against ARG1 (1:800, Abcam, Inc., Boston, MA, USA), an antibody against CD206 (1:1200, Abcam, Inc., Boston, MA, USA), or an antibody against β-actin (1:1200, Cell Signaling Technology, Inc., Danvers, MA, USA), then washed and incubated with horseradish peroxidase-coupled secondary antibodies for 2 h at 25 °C. The protein bands were detected by chemiluminescence (BioRad, Inc., Hercules, CA, USA), and their optical density was measured using Quantity One software (BioRad, Inc., Hercules, CA, USA). Relative protein levels were normalized to β-actin.