Ecl plus chemiluminescent reagent

Cells were homogenized and lysed with RIPA buffer (Thermo Scientific, MA, USA) containing a 1%phenylmethylsulfonyl fluoride (PMSF) and 1% protein phosphatase inhibitor mixture (Applygen, Beijing, China). The extracted proteins were harvested by centrifugation (12,000×g, 15 min, 4°C). Protein concentration was determined using the bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, MA, USA). Twenty micrograms of the extracted protein from each sample was subjected to electrophoresis in 8–12% SDS-PAGE depending on the molecular weight of the protein. Proteins in the gels were transferred onto polyvinylidene difluoride (PVDF) membranes (EDM Millipore, MA, USA) for Western blot analysis with the indicated antibodies. The sources of the antibodies and dilutions are as follows: anti-IBA1 (1:1000), anti-pERK (1:1000), anti-ERK (1:1000), and anti-GFAP (1:1000) were obtained from Cell Signaling Technology (Beverly, MA, USA); anti-β-actin (1:1000), anti-IL-6 (1:1000), anti-TNF-α (1:1000) and anti-IL-1β (1:1000), were all purchased from Santa Cruz Biotechnology (CA,USA). The ECL Plus chemiluminescent reagent (Thermo Scientific, MA, USA) was used to visualize the proteins. Protein bands were quantitated by ImageJ (NIH, USA).