Focal ischemia was induced by permanent occlusion of the left middle cerebral artery (MCA) under isoflurane anesthesia. Briefly, a skin incision was made between the orbit and the ear. Under an operating microscope, the temporal muscle was divided to locate the distal course of the left MCA through the translucent skull. A small burr-hole craniotomy was performed with a microdrill and the exposed MCA was coagulated using a bipolar pencil. Permanent occlusion was ensured by physical division. Animals were randomly divided into groups and received a single dose of 1, 5 and 15 mg/kg of purmorphamine (PUR) or VEH (PEG-400: ethanol: water (40 : 20 : 40) i.v. or i.p. at 6 h after MCAO. SHAM-operated animals were injected with VEH. CyP (10 mg/kg) was administered 40 min before PUR.

Smo binding assays were conducted with BODIPY-cyclopamine and Smo-overexpressing cells using CMV promoter-based, SV40 origin-containing expression constructs for Smo-Myc3, the deletion mutant Smo.Delta.CRD (deletion of amino acids 68 to 182), and Smo.Delta.CT (deletion of amino acids 556 to 793). HEK 293T cells were grown on poly-D-lysine-treated glass coverslips in 12-well plates until 70% confluency and then transfected with the appropriate expression construct (0.5 ug/well) using FuGene 6 according the manufacturer’s protocols. Two days after transfection, the HEK 293T cells were incubated with DMEM containing 0.5% bovine calf serum, 5 nM BODIPY-cyclopamine, and varying concentrations of purmorphamine (0, 1.5, or 5 uM) (1 mL/well) for 1 h at 37 °C. The Smo-overexpressing cells were then washed with 1X PBS buffer (1 mL/well), mounted with DAPI-containing medium, and visualized using a Leica DM4500B fluorescence microscope. For binding assays using fixed cells, the Smo-overexpressing HEK 293T cells were fixed with 3% paraformaldehyde in 1X PBS buffer for 10 min at room temperature (1 mL/well), treated with 1X PBS containing 10 mM glycine and 0.2% sodium azide for 5 min (1 mL/well), washed with 1X PBS buffer (1 mL/well), and treated with the compound-containing media described above for 4 h at room temperature.