Mouse anti rat β actin monoclonal antibody

The harvested cells were lysed in lysis buffer on ice and the cell extracts were clarified by centrifugation (at 9,500 × g). The protein concentrations were determined by spectrophotometry using the Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Aliquots containing 30 µg of protein were separated by SDS-polyacrylamide gel electrophoresis on a 10% gel, and the separated proteins were blotted onto polyvinylidene difluoride membranes in a wet transfer unit (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% non-fat dry milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with following primary antibodies: rabbit anti-rat GPR91 polyclonal antibody (1:1,000), rabbit anti-rat p-ERK1/2 monoclonal antibody (1:3,000), rabbit anti-rat ERK1/2 monoclonal antibody (1:3,000; #4695), rabbit anti-rat p-JNK monoclonal antibody (1:1,000), rabbit anti-rat JNK monoclonal antibody (1:1,000; #9252), rabbit anti-rat p-p38 monoclonal antibody (1:1,000), rabbit anti-rat p38 monoclonal antibody (1:1,000; #9212), goat anti-rat COX-2 polyclonal antibody (1:200; sc-1745; Santa Cruz Biotechnology, Dallas, TX, USA) and mouse anti-rat β-actin monoclonal antibody (1:5,000; A5441; Sigma-Aldrich, St. Louis, MO, USA). The bands were visualized using an enhanced ECL detection kit (Pierce Biotechnology, Rockford, IL, USA).