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After the cells reached 80% confluence, sample preparation for western blotting was performed using lysis buffer [50 mmol/l HEPES, 50 mmol/l NaCl, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1.5 mmol/l MgCl2, 1 mmol/l EDTA, 10 mmol/l sodium pyrophosphate, 1 mmol/l Na3VO4, 100 mmol/l NaF, 30 mmol/l 2-(p-nitrophenyl) phosphate, 1 mmol/l phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin and 10 mg/ml aprotinin (pH 7.4)] as previously described (39)

PMID 17244620

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