Protein a or g agarose sepharose bead slurry

We performed coimmunoprecipitation with rat brains as described previously [22 (link)]. After rats were anesthetized and decapitated, brains were removed. The cerebellum was homogenized in cold isotonic sucrose homogenization buffer containing 0.32 M sucrose, 10 mM HEPES, pH 7.4, 2 mM ethylenediaminetetraacetic acid (EDTA), and a protease/phosphatase inhibitor cocktail (Thermo Scientific, Rochester, NY). Homogenates were centrifuged at 800×g for 10 min at 4 °C. P2 pellets (synaptosomal fraction) were obtained by centrifuging the supernatant for 15 min (10,000×g) at 4 °C. Washed P2 was resuspended in the sucrose homogenization buffer containing Triton X-100 (0.5 %, v/v). The suspension was lysed and incubated with gentle rotation for 20 min at 4 °C. Samples were centrifuged for 20 min at 32,000×g to yield the pellet enriched with synaptic membranes. These pellets were solubilized in sucrose-Triton buffer containing 1 % sodium deoxycholate and a protease/phosphatase inhibitor cocktail for 1 h at 4 °C. Solubilized proteins were incubated with a rabbit antibody against ERK1/2 or mGluR1a. The complex was precipitated with 50 % protein A or G agarose/sepharose bead slurry (GE Healthcare). Proteins were separated on Novex 4–12 % gels and probed with a mouse antibody against ERK1/2 or mGluR1a.

Immunoprecipitation procedures have been described previously (Jin et al., 2013 (link); Mao and Wang, 2015 (link)). Briefly, for Fyn and Src immunoprecipitation, striatal tissue was homogenized in a RIPA lysis buffer. Samples were centrifuged at 800 g (10 min, 4°C) to remove insoluble materials. The supernatant was used for Fyn and Src immunoprecipitation. For phosphotyrosine protein immunoprecipitation, the P2 pellet was prepared from striatal tissue according to the procedures described above. The P2 pellet was solubilized in SHB containing 1% sodium deoxycholate for 1 h at 4°C. Solubilized proteins were used for phosphotyrosine protein immunoprecipitation. An equal amount of proteins (300–500 μg) was used for immunoprecipitation. Proteins were incubated with a mouse antibody against Src (2 μg), Fyn (3 μg), or phosphotyrosine (pTyr, 6 μg). The protein complex was precipitated with 50% protein A or G agarose/sepharose bead slurry (GE Healthcare). Precipitated proteins were separated on Novex 4–12% gels and probed with a rabbit antibody against Src, Fyn, phospho-Src family at Y416 (pan pY416), mGluR5, or pTyr. Horseradish peroxidase-conjugated secondary antibodies were used to visualize proteins.

P2 pellets (synaptosomal fraction) were prepared as described above and were solubilized in SHB containing Triton X-100 (0.5%, v/v) and 1% sodium deoxycholate for 1 h at 4°C. Solubilized proteins were incubated with a mouse antibody against Src or Fyn. The complex was precipitated with 50% protein A or G agarose/sepharose bead slurry (GE Healthcare). Proteins were separated on Novex 4-12% gels and probed with a rabbit antibody against Src, Fyn, or phospho-Src family at Y416 (pan pY416). HRP-conjugated secondary antibodies and enhanced chemiluminescence were used to visualize proteins.