NIH-3T3 cells were incubated at 37°C for 7h with vehicle control (0 µM) and 5 µM of jervine (ab120815) in DMSO. Decreased expression of smoothened (ab38686) in NIH-3T3 cells correlates with an increase in jervine concentration, as described in literature.Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with ab38686 at 1 µg /ml and ab8227 at 1 µg /ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.