Hybond enhanced chemiluminescence ecl nitrocellulose membrane

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.

BV2 cells were harvested and pelleted at 200 ×g for 3 min, followed by washing with phosphate-buffered saline (PBS). Cells were lysed with 20 mM Tris-HCl buffer (pH 7.4) containing protease inhibitors (0.1 mM phenylmethanesulfonyl fluoride, 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/mL chymostatin). Protein concentration was determined using the Lowry protein assay kit (P5626; Sigma). An equal amount of protein from each sample was resolved using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane from Bio-Rad (Hercules, CA, USA). The membrane was blocked with 5% skimmed milk and incubated with anti-HO-1 (1 : 1000 dilution), anti-iNOS (1 : 500 dilution), anti-COX-2 (1 : 1000 dilution), or anti-β-actin (1 : 1000 dilution) primary antibodies at 4°C overnight. The immunoreactive bands were visualized using a horseradish peroxidase-conjugated secondary antibody (1 : 1000 dilution) followed by ECL detection from Amersham Pharmacia Biotech (Piscataway, NJ, USA) and were quantitated using Image Gauge v3.12 software from Fujifilm (Tokyo, Japan).

Human liver-derived HepG2 cellswere harvested and pelleted by centrifugation at 200× g for 3 min. Then, 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 PMSF, 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., St. Louis, MO, USA). Thirty microgram of protein from each sample was resolved by 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 from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Cell Signaling, MA, USA) and a horseradish peroxidase-conjugated secondary antibody followed by ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA).