2.9 ELISA

Smooth Muscle Cells were infected with AdGFP or AdSmad3 and treated as previously described. At 24 hrs, conditioned media was collected and incubated overnight in a 96 well plate. Positive controls of β-catenin Ab and HRP-Rabbit Ab were utilized for positive controls. The plate was blocked in 2% BSA/PBS for 1 hr, and then incubated with primary antibody overnight at 4 °C. Antibodies to Wnts 5a (1:500, Sigma Aldrich, St Louis MO), Wnt9a (1:1000, Santa Cruz, Dallas TX), and Wnt11 (1:1000, Thermo Scientific, Rockford IL) were utilized. Plates were washed and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 4 h at room temperature. Afterwards, plates were washed and bound antibody was detected by Thermo Fisher West Pico Chemiluminescent Substrate (Waltham, MA) and analyzed using the Flexstation 3 plate reader (Molecular Devices, Sunnyville CA).