Anti human cox 2

Cells were washed with ice-cold phosphate buffered saline and incubated in extraction buffer (20 mM Tris-Cl [pH 7.4], 100 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 5 mM MgCI₂, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM pepstatin A, 0.1 mM antipain, 0.1 mM chemostatin, 0.2 mM leupeptin, 10 μg aprotinin, 0.5 mg/mL soybean trypsin inhibitor, and 1 mM benzamidine) on ice for 15 minutes. Equal amounts of total cell protein (100 μg) were loaded onto 10% sodium dodecyl sulfate–polyacrylamide denaturing gels and transferred to nitrocellulose membranes. The blots were then probed with anti-human COX-2, anti-Smad2, and anti-Smad3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were also incubated with antibody specific for α-tubulin (Sigma Chemical Co., St. Louis, MO) as a loading control. Antibody binding was detected using an ECL system (Amersham Pharmacia Biotech, Piscataway, NJ).