Anti cadherin 11

P14 forebrain and transfected and untransfected N2a cells were homogenized in lysis buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl and 1% Triton X-100 supplemented with PMSF (Cell Signaling Technologies catalog #8553S) and protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific catalog #78442). Protein extract (0.5 mg, standardized to 1 mg/ml) was precleared with 50 μl Protein-G Sepharose four Fast Flow (Millipore Sigma catalog #GE17-0618-01) or rProtein A Sepharose Fast Flow (Millipore Sigma catalog #GE17-1279-01) for 1 h at 4°C. Precleared supernatant was incubated with 2 μg anti-cadherin-8 (DSHB catalog #CAD8-1), 7 μg anti-cadherin-11 (Thermo Fisher Scientific catalog #32-1700) or 4 μg anti-HA antibodies (Millipore Sigma catalog #H6908), and mouse or rabbit IgGs for 2 h at 4°C. Samples were precipitated with 50 μl of preequilibrated Protein-G Sepharose four Fast Flow or rProtein A Sepharose Fast Flow for 1 h at 4°C with gentle mixing. Immunoprecipitates were washed three times in lysis buffer and eluted by boiling in 50 μl sample buffer. Overexpressed myc-flag-tagged cadherin-8 was immunoprecipitated using the Pierce c-My-tag IP/Co-IP kit according to the manufacturer’s protocol (Thermo Fisher Scientific catalog #23620). Co-immunoprecipitated proteins were determined by Western blot analysis.