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Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain

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2.4 Western blot analyses Whole cell protein lysates were prepared by lysis in radio-immunoprecipitation assay buffer (150 mM NaCl, 2 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS, 50 mM Tris pH 8.0, 1 mM PMSF, 1 g/ml aproptinin, 1 g/ml pepstatin, 1 g/ml leupeptin, 1 mM NaF, 1 mM sodium orthovanadate, 0.1 mM sodium molybdate, 10 mM sodium pyrophosphate) at a density of 4 10 7 cells/ml on ice, followed by addition of equal volumes of Laemmli protein sample buffer (60 mM TrisCl pH 6.8, 2 % SDS, 10 % glycerol, 5 % -mercaptoethanol, 0.01 % bromophenol blue) and heated to 95 C for 5 min prior to separation on SDS-PAGE gels and transfer to 0.45 m nitrocellulose membranes

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