Co-immunoprecipitation assay
Hippocampi from wild-type C57BL/6J mice were isolated and lysed in RIPA lysis buffer containing 1% v/v Triton X-100, 0.2% w/v SDS, 1% w/v sodium deoxycholate, 50 mM Tris-HCl (pH 7.6), 5 mM EDTA, 150 mM NaCl, 50 mM NaF, supplemented with antipain, leupeptin, benzamidine (1 μg/ml each), complete EDTA-free inhibitors, 1 mM PMSF and 1 mM sodium orthovanadate. Approximately 800 μg of the clarified cell lysates were incubated with an LC3B monoclonal antibody (2 μg) overnight at 4 °C. Normal rabbit serum (NRS) was used as control. Immune complexes were recovered on protein A agarose beads for 3 hours at 4 °C, washed extensively with buffer consisted of 25 mM Tris-HCl (pH 7.4), 300 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 % Triton X-100, 0.2 mM PMSF and 0.2 mM Na3VO4, subjected to SDS-PAGE and transferred onto polyvinylidene (PVDF) membranes. Immunoprecipitation of cell lysate proteins was verified by immunoblotting using the appropriate antibodies.