KCCs phosphorylated at the KCC2 Thr906 and Thr1007 equivalent residue were immunoprecipitated from clarified hippocampal and cortical culture lysates (centrifuged at 16,000g at 4°C for 20 min) using phosphorylation site–specific antibody coupled to protein G–Sepharose as described (31). The phosphorylation site–specific antibody was coupled with protein G–Sepharose at a ratio of 1 mg of antibody per 1 ml of beads in the presence of lysate (20 μg/ml) to which the corresponding nonphosphorylated peptide had been added. Two milligrams of clarified cell lysate was incubated with 15 μg of antibody conjugated to 15 μl of protein G–Sepharose for 2 hours at 4°C with gentle agitation. Beads were washed three times with 1 ml of lysis buffer containing 0.15 M NaCl and twice with 1 ml of buffer A. Bound proteins were eluted with 1× LDS sample buffer.

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