2.11. Statin treatment in cultured mammalian cells

RAW264.7 cells were seeded at a density of 300,000 cells in six-well dishes. The media were replaced the next day, and fluvastatin, simvastatin, DMSO, and mevalonolactone were added at the indicated concentrations. At 72 h post-treatment, the cells were washed twice with ice-cold PBS and lysed in 150 μl of RIPA/SDS buffer (50 mM Tris pH 8, 150 mM NaCl, 5 mM EDTA, 1% v/v NP-40, 0.5% w/v deoxycholic acid, 0.1% w/v SDS, 10 mM NaF, 0.1 mM PMSF, and Complete Protease Inhibitor cocktail tablets). The total protein concentrations were determined using a Bradford Assay (Bio-Rad) and 40 μl of the samples were diluted in 5 x Laemmli sample buffer and resolved by SDS-PAGE. Next, Western blotting membranes were probed with antibodies against rabbit polyclonal phospho-p38 (Cell Signaling, Cat. #9211), mouse monoclonal p38 (Abcam, Cat. #AB31828), and mouse monoclonal anti-actin C4 (MP Biomedicals, Cat. # 8691002), all at 1:1000 dilution, and goat polyclonal anti-COX-2 (Santa Cruz Biotechnology, Cat. #SC-1745) at 1:400 dilution. Horseradish peroxidase-conjugated bovine anti-goat IgG, goat anti-rabbit IgG, and goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) were used at a dilution of 1:10,000. The proteins were visualized using WesternBright ECL (Advansta) in an Amersham Imager 600 and quantified by ImageQuant TL software.