To perform co-immunoprecipitation with neural tissues, the CNS of 3rd-instar larvae (~120 per experimental condition) were dissected out and placed in ice-cold 1x PBS containing 2 mM sodium vanadate. After a pulse of centrifugation, larval CNS were isolated and then lysed on ice with the lysis buffer (50 mM Tris-HCl/pH 7.4, 150 mM NaCl, 2 mM sodium vanadate, 10 mM sodium fluo-ride, 1% Triton X-100, 10% glycerol, 10 mM imidazole and 0.5 mM phenylmethylsulfonyl fluoride). Lysates were centrifuged at 15,000 g for 20 min at 4°C. The resulting supernatants were either saved as inputs or incubated with magnetic beads conjugated with appropriate antibodies for 4 hours at 4°C. After washing once with the lysis buffer, twice with lysis buffer containing 0.1% deoxycholate, and 3 times with lysis buffer lacking Triton X-100, the immunoprecipitates and total lysates were resolved on 7.5% SDS-PAGE gels followed by western blotting as previously described (Kim et al., 2013 (link)). Protein samples were transferred to nitrocellulose membranes and detected by chemiluminescence (Catalog# 32106, Pierce ECL Western Blotting Substrate) with either a BIO-RAD ChemiDoc Touch Imaging system or a Kodak X-OMAT 2000 film processor.
The procedure of co-immunoprecipitation with lysates from S2 cells was similar except that cultured S2 cells were re-suspended in ice-cold 1x PBS before lysis.
The procedure of co-immunoprecipitation with lysates from S2 cells was similar except that cultured S2 cells were re-suspended in ice-cold 1x PBS before lysis.