An SCC9, SCC25, CAL27, and FaDu cell monolayer (105 cells in a 6-cm tissue culture dish) was rapidly rinsed twice with ice-cold phosphate-buffered saline and lysed in 0.5 mL of ice-cold lysis buffer. The lysis buffer contained 1% phenol-containing detergent (NP-40), 0.1% sodium dodecyl sulfate, 150-mmol/L sodium chloride, 50-mmol/L Tris at a pH of 7.4, 10-mmol/L edetic acid, 10-mmol/L p-nitrophenolphosphate, 250-U/L aprotinin, 40-μg/mL leupeptin, 1-mmol/L phenylmethylsulfonyl fluoride, 1-mmol/L sodium orthovanadate, 10-mmol/L sodium fluoride, and 40-mmol/L β-glycerolphosphate. The lysates were centrifuged at 14 000 rpm at 4°C for 20 minutes, and the supernatants were collected. Aliquots of supernatants containing 20 μg of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel in 10% gels and transferred to nitrocellulose membranes. After blocking nonspecific binding by incubation with 5% bovine serum albumin in Tris-buffered saline (Bio-Rad Laboratories, San Diego, California), the membranes were incubated with anti–COX-1 or anti–COX-2 (Santa Cruz Biotechnologies, Santa Cruz, California) as primary antibodies. Bound antigen was visualized with the ECL Western blotting analysis system (Amersham Life Sciences, Buckinghamshire, England). The density of bands was measured by Quantity One Basic Software (Bio-Rad Laboratories).