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For the sample at t 0 , an aliquot of UBE2L3~UB* was diluted to 0.4 µM in 25 mM HEPES and 150 mM NaCl, pH 7.5, and a sample was immediately removed and quenched with 2 × SDS–PAGE sample buffer (100 mM Tris-HCl, 20% glycerol, 30 mM EDTA and 4% SDS)