Protein a and g agarose sepharose bead slurry

The rat cerebellum was removed after anesthesia and decapitation and was homogenized in cold isotonic homogenization buffer containing 0.32 m sucrose, 10 mm HEPES, pH 7.4, 2 mm EDTA, and a protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific). Lysates were then processed at 4°C. After centrifugation (800 × g, 10 min), the supernatant was centrifuged again (10,000 × g, 15 min) to get P2 synaptosomal pellets. P2 pellets were solubilized in the buffer containing Triton X-100 (0.5%, v/v), 1% sodium deoxycholate, and a protease/phosphatase inhibitor cocktail for 1 h. After centrifugation, the solubilized supernatant was used for coimmunoprecipitation. HEK293T cells were lysed and solubilized in RIPA buffer (Sigma-Aldrich). Solubilized supernatant proteins after centrifugation were used for coimmunoprecipitation. Solubilized samples were incubated with a rabbit or mouse antibody. The complex was precipitated with 50% protein A and G agarose/Sepharose bead slurry (GE Healthcare). Proteins were separated on Novex 4–12% gels and probed with immunoblotting with a mouse or rabbit antibody if a rabbit or mouse antibody was used in immunoprecipitation, respectively.