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Western Blotting Approximately 50100 mg fresh-frozen tumors were cut into small pieces, partially thawed, and washed in ice-cold PBS containing 1 mM phenylmethylsulfonyl fluoride, incubated for 30 min at 4 C in ice-cold lysis buffer (20 mM HEPES, 150 mM NaCl, 5 mM EDTA, 3 mM EGTA, and 4 mg/ml dodecyl -d-maltoside (pH 7.6)) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 g/ml soybean trypsin inhibitor, 10 g/ml leupeptin, 10 g/ml aprotinin, and 50 g/ml bacitracin), and homogenized in a tissue lyser Miller Mix for 2 min at 20 U/s