Protein Extraction and Immunoblotting Protocol

For protein extraction, pooled mesenteric arteries were lysed in a buffer containing 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.5), 2 mmol/L EDTA, 1% v/v NP-40, 0.5% w/v deoxycholate, 10 mmol/L NaF, 10 mM sodium pyrophosphate, 2 mmol/L PMSF, 2 g/mL leupeptin, and 2 g/mL aprotinin, pH 7.4. Lysates were incubated on ice for 15 min and then centrifuged at 38,000× g for 30 min at 4 °C to collect the supernatant. Protein concentration was measured using a dye-binding protein assay kit (Bio-Rad) and reading to the spectrophotometer at a wavelength of 595 nm. Immunoblotting was performed as previously described [25 (link)], using the following antibodies: anti-bactin (Abcam, ab49900; mouse monoclonal, 1:4000), anti-phospho-ERK1/2 (Santa Cruz, sc-136521; mouse monoclonal 1:800), anti-AT1 receptor (Santa Cruz, sc-57036; mouse monoclonal 1:1000), anti-Rac1-GTPγ (STA-401-1, Cells Biolab Inc., 1:800). Secondary antibodies (1:3000) were purchased from Amersham Life Sciences (GE Healthcare). Bands were visualized with enhanced chemiluminescence (ECL, Amersham Life Sciences), according to the manufacturer’s instructions. Immunoblotting data were analysed using ImageJ software (developed by Wayne Rasband, NIH, Bethesda, MD, USA) to determine the density of the bands.