The membranes were counterstained using corresponding donkey anti-guinea pig (1 : 5000; Jackson Immunoresearch, West Grove, PA, USA), goat anti-rabbit or anti-mouse (both 1 : 3000; Bio-Rad Laboratories, Hercules, CA, USA) horseradish peroxidase conjugates. For stripping between the immunoblot procedures, membranes were rinsed and incubated in Restore Western Blot Stripping Buffer (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. For visualization of the proteins, the membranes were exposed to the enhanced chemiluminescence detection system Lumigen PS-3 (1 : 40; GE Healthcare, Buckinghamshire, UK). No immunopositive bands were observed

when immunoblotting was performed with anti-CB1 antibodies pre-absorbed with the antigene peptide (5 μg/mL; Frontier Science, Japan). For immunoprecipitation, ~2.0 mg of total protein from mouse embryo (E16.5) brain mitochondrial fractions

(prepared RAD001 as above) was incubated overnight at +4 °C with 3 μL of made-in-guinea pig anti-CB1 sera (Frontier Science, Japan). Thirty microliters of a 1 : 1 slurry of protein A-sepharose (GE Healthcare, Buckinghamshire, UK) in phosphate-buffered saline was then added and antibody-bound protein was collected during a 2-h incubation at +4 °C. GDC-0449 in vivo The Sepharose beads were washed four times in 500 μL phosphate-buffered saline containing protease inhibitor cocktail (1 : 500; Calbiochem, La Jolla, CA, USA). The beads and bound protein were loaded in mini gel and separated using electrophoresis as above. The gel was then stained with SimplyBlue colloidal Coomassie (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The ~40-kDa band was cut from the gel and destained

in three washes of acetic acid : methanol : H2O (10 : 50 : 40) solution. The sample was submitted for in-gel tryptic digestion, followed by liquid chromatography, quadrupole/time-of-flight tandem mass spectrometry and peptide mass database searching (Keck Facility, Yale University, New Haven, CT, USA). Mouse neuroblastoma 2A cells were cultured in Dulbecco’s D-MEM/F12 medium containing 9% fetal bovine serum (all from Sigma-Aldrich, St Louis, MO, USA). For transfections, we cloned full-length SLP-2 from E14.5 embryo brain cDNA into pIRES2-EGFP (Clontech, Mountain View, CA, USA); transfections with pEGFP C-X-C chemokine receptor type 7 (CXCR-7) (Clontech, Mountain View, CA, USA) were used as negative controls. Newly passaged cells at about 70–80% confluency were starved of serum overnight and transfected with 5 μg SLP-2 DNA using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guidelines. After 24 h, cells were washed in phosphate-buffered saline, and immediately scraped and lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing protease (Roche, Indianapolis, IN, USA) and phosphatase (Sigma-Aldrich, St Louis, MO, USA) inhibitor cocktails.