Immunoprecipitation and immunoblot

Immunoprecipitation and immunoblot were performed as described (Toriyama et al., 2006). For immunoprecipitation with HEK293T cells, cell lysates were prepared using NP40 lysis buffer (0.5% NP-40, 20 mM HEPES pH 7.5, 3 mM MgCl₂, 100 mM NaCl, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 0.01 mM leupeptin, 1 × PhosStop). The supernatants of cell lysates were incubated with 25 μl (bed volume) of anti-FLAG M2 gel (RRID:AB_10063035) overnight at 4°C. The anti-FLAG M2 gels were washed three times with wash buffer (0.1% Tween 20, 20 mM HEPES pH 7.5, 3 mM MgCl₂, 100 mM NaCl, 1 mM EGTA, 1 mM DTT) and once with TED buffer. To elute immunocomplexes, the gels were incubated with 25 μl of FLAG peptide (400 μg/ml) for 2 hr at 4°C. The immunocomplexes were analyzed by immunoblot.

For immunoprecipitation with cultured neurons, after netrin-1 (4.4 nM) stimulation for 1 hr, cell lysates were prepared with NP40-Triton lysis buffer (0.5% NP-40, 1% Triton X-100, 20 mM HEPES pH 7.5, 3 mM MgCl₂, 100 mM NaCl, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 0.01 mM leupeptin, 1 × PhosStop). The supernatants of the lysates were incubated with antibodies overnight at 4°C, and immunocomplexes were then precipitated with protein G-Sepharose 4B. After washing the beads with wash buffer (0.1% Tween 20, 20 mM HEPES pH 7.5, 3 mM MgCl₂, 100 mM NaCl, 1 mM EGTA, 1 mM DTT), immunocomplexes were analyzed by immunoblot.