Anti at1 receptor

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.

The AT1 receptor, CML and RAGE contents in the aortic arch wall were evaluated by immunofluorescence. Specimens were mounted on glass slides with 3-aminopropyltriethoxysilane (Sigma Chemical, Co.), washed in phosphate-buffered saline (PBS) and blocked with 5% bovine serum albumin in PBS for 30 min at room temperature. The slices were incubated overnight at 4°C with rabbit polyclonal anti-AT1 receptor (1:40; Santa Cruz Biotechnology, Inc., Cat # SC1173, Dallas, TX, USA), rabbit polyclonal anti-CML (1:40; Immundiagnostik AG, Cat # 1109, Bensheim, Germany) and rabbit polyclonal anti-RAGE (1:50; Santa Cruz Biotechnology, Inc., Cat # SC 5663, Dallas, TX, USA) antibodies diluted in PBS. The sections were next washed in PBS with 0.05% Tween-20 and incubated for 90 min at room temperature with Alexa 488-conjugated goat anti-rabbit IgG antibody (Life Technologies, Cat # A11008, Grand Island, NY, USA) diluted at 1:200 in a PBS solution containing 0.006% Evans blue dye. Finally, the samples were mounted in buffered glycerol and analyzed using fluorescence microscopy (Olympus BX51, Olympus, Co., Tokyo, Japan). For negative and autofluorescence controls, sections were incubated with PBS instead of the specific antibody.