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In vitro reconstitution of Rex with NAD+ and NADH.
(A) The UV-Vis spectra of rRex after purification (light line). Incubation of rRex with 20-fold excess NADH (dark line) overnight at 4°C generates a different UV-Vis spectrum with a spectral peak at 340 nm, indicative of NADH binding to rRex. (B) SDS-PAGE after rRex reconstitution with either NAD+ or NADH verifies the presence of rRex. (C) EMSA analysis with the adhE promoter with NAD+-loaded (lane 2) and NADH-loaded (lane 3) rRex, or rRex as a negative control (lane 1). No additional NAD+ or NADH was included in the EMSA reactions.