dLKR/SDH is recruited to the dronc promoter and localizes to the cytoplasm and nucleus. (A) Nuclear extracts from cells treated with either ethanol (−) or 10 μM ecdysone (Ecd +) for 2 h were incubated with double-stranded biotin-tagged wild-type (WT) or mutated (mut) oligonucleotides corresponding to the dronc EcR/Usp BE and pulled down with streptavidin beads. Proteins were separated by SDS-PAGE and stained, and protein bands were isolated. Part of the pull-down was blotted with EcR-B1 antibody. (B) LKR converts lysine to saccharopine by oxidizing NADH to NAD⁺ and condensing α-ketoglutarate molecule onto the amino group. (C) GST, GST-LKR, GST-SDH, and GST-SDH deletion mutants containing the indicated residues (618–928 or 803–928) were used in pull-down experiments with ³⁵S-labeled EcR/Usp in the presence of ethanol control (−) or ecdysone (+). Half of the reaction was used in SDS-PAGE and stained with Coomassie Brilliant blue. (D) Lysates from cells treated with ethanol (−) or ecdysone (Ecd +) for 4 h were immunoprecipitated (IP) with EcR-B1 or a control (N27A1) antibody and blotted with dLKR/SDH and EcR-B1 antibodies. The bottom panel is a dLKR/SDH immunoblot of the lysates used in immunoprecipitation. (E) 200 ng/ml of affinity-purified dLKR/SDH antibody was used to blot extracts from l(2)mbn cells treated with a control (Ndfip2) dsRNA or dLKR/SDH dsRNA. Lysates from either wild-type, homozygous (Df/Df), or heterozygous (Df/+) Drosophila carrying a deficiency that removes the dLKR/SDH locus were probed with the dLKR/SDH antibody. This antibody cross reacts with a nonspecific protein (asterisks) of ∼65 kD (here and elsewhere in immunoblots). dLKR/SDH protein bands are indicated by arrows. The same filter was blotted with heterochromatin protein 1 (HP1). (F) l(2)mbn cells were treated with ethanol (0) or ecdysone as indicated, and nuclear and cytoplasmic fractions were blotted with the indicated antibodies.