Lowry protein assay kit

BV2 cells were lysed in 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM PMSF, 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/mL chymostatin). The protein concentration was determined using the Lowry protein assay kit (P5626; Sigma). An equal amount of protein from each sample was resolved using 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to the Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked using 5% skim milk and subsequently incubated with the primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and horseradish peroxidase-conjugated secondary antibody, followed by ECL detection (Amersham Corp., Arlington Heights, IL).

RAW 264.7 macrophages were harvested and pelleted by using centrifugation at 200× g for 3 min. Then, the cells were washed with phosphate-buffered saline and lysed in 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM phenylmethanesulfonyl fluoride, 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/mL chymostatin). Protein concentration was determined using a Lowry protein assay kit (Sigma Chemical Co.). Thirty micrograms of protein from each sample were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then electrophoretically transferred onto a Hybond enhanced chemiluminescence nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skimmed milk and sequentially incubated with the primary antibody (Santa Cruz Biotechnology and Cell Signaling Technology) and a horseradish peroxidase-conjugated secondary antibody, and then subjected to enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Western blotting was performed as follows. After harvesting, the cells were pelleted by centrifugation for 3 min. Next, the cells were washed with PBS and lysed in 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM phenylmethanesulfonyl fluoride, 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/mL chymostatin). Protein concentration was determined using a Lowry protein assay kit (Sigma-Aldrich). Protein (30 mg) from each sample was resolved using 7.5% and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoretically transferred onto a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skimmed milk and sequentially incubated with the primary antibody and a horseradish peroxidase-conjugated secondary antibody, followed by ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The following antibodies were used: IκBα, NF-κB p65, β-actin (Cell Signaling Technology, Danvers, MA, USA), HO-1 (Merck), iNOS (Cayman Chemical, Ann Arbor, MI, USA), PCNA (Millipore, Middlesex, MA, USA), and Anti-COX2/cyclooxygenase 2 (Abcam, Cambridge, UK). In addition, the cytosolic and nuclear fractions were extracted using the Cayman Nuclear Extraction Kit (Cayman Chemical), and each fraction was lysed according to the manufacturer’s instructions.