MLO-Y4 osteocytes or HEK293 cells were washed with ice-cold PBS, lysed with a lysis buffer [25 mM Hepes·Na (pH 7.2), 150 mM CH3COOK, 2 mM EDTA, 0.1% NP-40, 10 mM NaF, 1 mM DTT, and protease inhibitor cocktail] (46), scraped, and collected in a tube. After 10-min incubation on ice, samples were cleared of cell debris by centrifugation at 13,000g for 10 min and incubated with specific antibodies. Immunoprecipitation was conducted using PureProteome Magna Protein A Magnetic Beads (Millipore, Billerica, MA, USA) according to the manufacturer’s instruction, and proteins were recovered from resultant immunoprecipitates by 1× SDS sample buffer and subjected to SDS–polyacrylamide gel electrophoresis followed by immunoblotting.

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