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We incubated the extract overnight at 4 C with antibodies to NDRG1 (1:400) or PLC1 (1:50) in NET-gel buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.25% gelatin, 0.02% sodium azide, 1 mM PMSF, 10 mg/ml leupeptin, and 10 mg/ml aprotinin), and, then, with Protein G Agarose (16-266

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