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For K48- or K63-TUBE pulldowns, cells were lysed in 150 L of lysis buffer [100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% Triton-X100, 10% glycerol] supplemented with 20 M MG132, 10 M PR619, 10 mM iodoacetamide, and a protease inhibitor cocktail (Nacalai Tesque) and were then extensively sonicated (Handy Sonic, TOMY Seiko)

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For example, the genes encoding transferrin receptor (TFRC), glucose transporters and the master antioxidant regulator NRF2 are closely associated with the pathological progression of AD (49)

The amplified DNA fragment was cloned into the EcoRI/HindIII site of the pET-26b ( + ) plasmid encoding a C-terminal histidine tag (Novagen)

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This instrumental breakthrough resulted in the widespread application of CD spectroscopy, making this method one of the major tools in protein structure investigation