Rho-activation assay

RhoA activity was determined using the G-LISA RhoA activation assay biochemical kit (Cytoskeleton, Inc). Active RhoA was assessed according to the manufacturer's instructions. In brief, first and second order mesenteric arteries were isolated and mounted in a pressure myograph. The pressure was increased step-wise up to 80 mmHg until the myogenic contraction was apparent. In case of Sm-12/13-KO and Sm-Larg-KO animals in which no myogenic contraction could be observed, arterial segments were pressurized step-wise up to 80 mmHg and RhoA activity was determined after 5 min. Arteries were immediately snap-frozen and lyzed and processed according to the manufacturer's instructions. 50 μl of the protein sample (0.5 μg/μl) from arterial segments were added to 96-well plates coated with the Rho binding domain of Rho effector protein and incubated at 4°C for 30 min under shaking. The plates were subsequently incubated with anti-RhoA antibody and secondary horseradish peroxidase-conjugated antibody for 45 min at room temperature. Active RhoA levels were determined by measuring absorbance at 490 nm using a microplate spectrophotometer.