Ecl western blot system

Aliquots of samples were subjected to SDS-PAGE using a 10% gel (for COX-2 and PGIS) or 15% gel (for mPGES-1) under reducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell, Hahnenstr, Germany) with a semidry blotter (Bio-Rad Laboratories, Hercules, CA). After blocking with 5% (w/v) skim milk in Tris-buffered saline (TBS) (pH 7.4) containing 0.05% (v/v) Tween-20 (TBS-Tween), the membranes were probed with the respective antibodies for 1.5 h (1:5000 dilution for anti-COX-2 antibodies (Santa Cruz Biotechnology, Dallas, TX), 1:2500 dilution for anti-mPGES-1 (Cayman Chemical, Ann Arbor, MI), anti-PGIS (Genway Biotech, SanDiego, CA) and anti-β-actin antibodies (Sigma, St. Louis, MO) in TBS-Tween). They were then incubated with horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G antibodies (1:5000 dilution in TBS-Tween) for 1 h, and visualized with the ECL western blot system (Perkin-Elmer Life Sciences, Boston, MA), as described previously6 (link)9 (link).

Co-immunoprecipitation assay and Western blot were performed as described [34 (link)]. Lysates were analyzed by immunoblot analysis using the specific antibodies as indicated in the text. Specific bands were detected with a horseradish peroxidase-conjugated antibody and revealed by an enhanced chemiluminescence (ECL) Western blot system (PerkinElmer, Waltham, MA).