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.