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After centrifugation and washing 35 times with wash buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM DTT), the protein-bound beads were loaded onto an SDS-PAGE gel for western blot analysis using antibodies against 6 His and GST

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