dLKR/SDH represses ecdysone receptor–mediated transcription. (A) 5 μg of recombinant active or heat-inactivated dLKR/SDH was incubated with 1 mM NADH, 2 mM α-ketoglutarate, and 14 mM lysine. Buffer without enzyme (−) was incubated for 4 h before measuring NADH levels. Two experiments were performed in triplicate. (B) 2.5 × 10⁶ cells were transfected with 1 μg luciferase reporter driven by the EcR BE of the Hsp 27 promoter (pEcR-Luc) and pIB dLKR/SDH (dLKR/SDH) in the presence of ethanol (−) or 10 μM ecdysone (Ecd +). Each experiment was performed in triplicate, and total DNA was made up to equal amounts using vector. 30 μg of lysate was assayed for luciferase activity. Data were derived from at least three experiments. (C) Same as in B, but the NADH domain of dLKR/SDH was mutated by substituting amino acids 221, 223, and 228 to alanine (dLKR/SDH mut). (D) Experiments performed as in B, but a luciferase reporter with the proximal promoter of dronc containing multiple BR-C BEs was used (BR-C Luc). Either empty expression vector pIE (−) or vector expressing BR-C (+) was cotransfected where indicated. (E) Experiments performed as in B, but dsRNA corresponding to mammalian Ndfip2 (cont) or dLKR/SDH was included with pEcR-Luc. Ethanol vehicle (−) or ecdysone (+) was included as shown. (F) Experiments performed as in D using the BR-C reporter (BR-C Luc) and the transcription factor BR-C overexpressed where indicated (+). dsRNA (RNAi) corresponding to Ndfip2 (cont) or dLKR/SDH was included where indicated. Error bars represent SD.