Antibody against inos

The dried aqueous extracts of seven QFY ingredients were manufactured by Nong's Pharmaceutical Ltd., Hong Kong. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin solution were purchased from Invitrogen (Carlsbad, CA, USA). Protein assay dye reagent concentrate was purchased from Bio-Rad (Hercules, CA, USA). Lipopolysaccharide (LPS), RIPA assay buffer, protease inhibitor cocktail, and anti-rabbit horseradish peroxidase- (HRP-) conjugated IgG secondary antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). An antibody against iNOS was purchased from Abcam (Cambridge, UK). Antibodies against COX-2 and GAPDH were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-mouse HRP-conjugated IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). An enhanced chemiluminescence (ECL) detection reagent was purchased from GE Healthcare (Uppsala, Sweden). Ginsenoside Rg3 with the purity of >98% was purchased from Nanjing Spring and Autumn Biological Engineering Company (Nanjing, China).

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.

Aspalatone was purchased from Cayman Chemical Company. Endothelial Cell Medium (ECM) was obtained from ScienCell™ Research Laboratories. PBS, penicillin/streptomycin solution, and Trypsin/EDTA were obtained from Invitrogen. FBS was obtained from Gemini Bio-Products. MTT was obtained from Sigma. RIPA buffer was obtained from Santa Cruz Biotechnology. Vascular Endothelial Growth Factor (VEGF-165) and antibodies against VCAM-1, ICAM-1, eNOS, and GAPDH were obtained from Cell Signaling Technologies. Antibody against iNOS was obtained from Abcam. CM-H₂DCFDA was obtained from Molecular Probes, Invitrogen. Malondialdehyde (MDA) detection kit was obtained from Oxis Research. Human Milliplex angiogenic cytokine multiplex kit was obtained from Millipore. All other reagents and chemicals used were of analytical grade and were obtained from Sigma.