4.9. Western Blot

Protein expression of p38 MAPK (total and phosphorylated), Keap1, Bach1, and beta-actin was determined by Western blot analysis in cultured endothelial cells and isolated aortas. Samples were homogenized in lysis buffer, and proteins were kept frozen in −80 °C until use. Proteins (30 μg) were separated by electrophoresis on 10% or 12% polyacrylamide gels, transferred to 0.22 μm nitrocellulose membranes, and blocked using 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) and 0.1% Tween 20 for 1 h. Primary antibodies were incubated overnight at 4 °C, as follows: anti-phospho p38 (phospho Thr180 + Tyr182) (1:1000; Cell Signaling 4511), anti-p38 (1:1000; Cell Signaling 9212), anti-Keap1 (1:1000; Abcam 66620), anti-Bach1 (1:1000; sc-271211), and anti-β-actin-peroxidase (1:10,000 dilution; Sigma-Aldrich). Protein bands were detected by chemiluminescence reaction (Luminata Forte, WBLUF0100, Merck-Millipore, Watford, UK), and the intensity of the bands was evaluated by densitometric analysis using ImageQuant software.