The cell culture supernatant was concentrated with Amicon ultracentrifugation filter units (Millipore, Massachusetts, USA) and precleaned by incubation with protein G-Sepharose (GE Healthcare Life Sciences, Buckinghamshire, UK) beads for 2 h at 4°C. The supernatant was incubated overnight with anti-CASK antibody (Santa Cruz, H-107) in a rotating mixer at 4°C. protein G-Sepharose beads were then added and the mixture was incubated for 2 h at 4°C. It was then centrifuged and the beads were washed. The complexes were recovered in 2× Laemmli buffer (BioRad, California, USA) and analyzed by SDS-PAGE.
For co-immunoprecipitation, cells (1.5 × 107) were lysed by incubation in ice-cold lysis buffer (25 mmol/L HEPES, 150 mmol/L NaCl) supplemented with 1% Brij 97 and a protease inhibitor cocktail (Thermo Fisher Scientific, Massachusetts, USA) for 1 h at 4°C. The lysate was centrifuged (10 min, 20000 × g), and 1 mL of the supernatant was then precleared by incubation with protein G-Sepharose for 2 h at 4°C. The precleared lysates were incubated overnight at 4°C with 1 μg antibody. They were then incubated with protein G-Sepharose for 2 h, and the immune complexes were washed five times in the lysis buffer. Immunoprecipitated proteins were analyzed by SDS-PAGE and western blotting.
For co-immunoprecipitation, cells (1.5 × 107) were lysed by incubation in ice-cold lysis buffer (25 mmol/L HEPES, 150 mmol/L NaCl) supplemented with 1% Brij 97 and a protease inhibitor cocktail (Thermo Fisher Scientific, Massachusetts, USA) for 1 h at 4°C. The lysate was centrifuged (10 min, 20000 × g), and 1 mL of the supernatant was then precleared by incubation with protein G-Sepharose for 2 h at 4°C. The precleared lysates were incubated overnight at 4°C with 1 μg antibody. They were then incubated with protein G-Sepharose for 2 h, and the immune complexes were washed five times in the lysis buffer. Immunoprecipitated proteins were analyzed by SDS-PAGE and western blotting.