Western blotting reagents

We used Western blot to determine the expression of CSE in carotid sinus. After experiment, the bilateral carotid sinus were rapidly removed and put in liquid nitrogen and then stored at -80 °C for further analysis. The tissue were homogenized in 100 μl lysing buffer and then centrifuged at 15,000 g for 20 min at 4 °C. We collected supernatant for protein assay. Bradford assay was used to determine the concentration of protein in tissue. The protein was denatured at 99 °C for 10 min. Then protein of 50 μg was loaded in each lane of SDS-PAGE gels. After electrophoresis, the protein was separated then transferred onto polyvinylidene fluoride (PVDF) membranes. The transferred PVDF membranes were blocked with 5 % skim milk in TBST (1.37 mmol/l NaCl, 200 mmol/l Tris, 0.05 % Tween-20, pH 7.5) for 1 h. The PVDF membranes were incubated with primary antibody (anti-mouse CSE polyclonal antibody, 1:500, Proteintech Biotechnology) overnight followed by appropriate secondary horseradish peroxidase-conjugated antibody (1:2000, Proteintech Biotechnology). Western blotting reagents (Millipore Corporation, Billerica, MA01821, USA) were used to detect the signal. The chemiluminescent signals obtained were recorded. We have used Photoshop to modulate the lightness of the photographies (Fig. 6).