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Then the immunoprecipitates were collected by centrifugation (12000 rpm, 4 min) and washed three times with cell lysis buffer (NP-40, Beyotime), then boiled for 5 min with the same volume of 2× loading buffer (62.5 mM Tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue)
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