Phosphatase inhibitors cocktail 2

Cells were resuspended in break buffer (20 mM HEPES pH 7.4, 0.5 mM MgCl₂, 0.13 M sucrose, 50 mM NaCl, 1 mM PMSF, phosphatase inhibitors cocktail 2 (Sigma) and protease inhibitor mixture (Roche)) and passed through a ball‐bearer homogeniser 20 times using 0.8 μm ball. An aliquot of the sample was reserved as the “total lysate”, and the remainder was centrifuged at 500 × g for 10 min at 4°C to pellet the nuclei and cell debris. The supernatant was ultracentrifuged at 150,000 × g for 1 h at 4°C (Beckman TLA‐55) to obtain the cytosol and membrane fractions. The membranes were washed twice with break buffer. The membrane fraction was resuspended in break buffer in equivalent volume to cytosol fraction. Sample buffer was added to each fraction, and analysis was performed by immunoblot.

Cells were collected and lysed by using IP lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM EGTA, 5 mM MgCl2, 10%glycerol and 0.2%NP-40), supplemented with protease inhibitor (Thermo Fisher Scientific), phosphatase inhibitors cocktail 2 (Sigma-Aldrich, P5726), and 1 mM 1,4-dithiothreitol (Sigma-Aldrich). Then cell lysate was centrifuged 20000g for 15 min at 4 °C, and the supernatant was incubated with primary antibody for 4 h at 4 °C. After that, the antibody-bound protein was subjected to incubate with Protein A/G Beads (ThermoFisher, 88802) for 2 h at 4 °C. After six washes with wash buffer (0.5 M Tris-HCl pH 7.4, 1.5 M NaCl), immunoprecipitated complexes were denatured with loading buffer (Fdbio science) for 10 min at 95 °C. The samples were then stored at −20 °C or ready for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

Brain homogenates were prepared as previously described^{59 (link)}. Briefly, 200 mg of frontal cortex were homogenized in 5 volumes of 20 mM Tris-HCl, pH 7.5, 140 mM NaCl, added with Complete Protease Inhibitors cocktail and Phosphatase Inhibitors Cocktail 2 (Sigma) using a manual Dounce homogenizer and ultracentrifuged at 100,000 xg for 1 hour at 4 °C. The supernatant was collected, aliquoted and stored at −80 °C as the S1fraction. The pellet was re-homogenized in 1% Chaps, 1% Deoxycholate, 0.2% SDS, 140 mM NaCl, 10 mM Tris-HCl, pH 7.5, added with Protease and Phosphatase Inhibitors and ultracentrifuged at 30,000 xg for 30 minutes at 4 °C. The supernatant was aliquoted and stored at −80 °C as the S2 fraction; the pellet was homogenized in 2% SDS, 20 mM Tris-HCl, pH 7.5, 140 mM NaCl and ultracentrifuged at 30,000 xg for 30 minutes at 4 °C. The supernatant was saved as the S3 fraction and stored at −80 °C; the pellet was extracted in 4% SDS, 8 M Urea (P3 fraction). The total proteins amount was measured in each fraction by BCA Protein Assay kit (Pierce). Immunodepletion was carried out by using Protein G Mag Sepharose beeds (GE Healthcare) and a mixture of 4G8 and 6E10 antibodies.