dLKR/SDH is recruited to the endogenous dronc and ark promoters. (A) For ChIP analyses, 10⁷ l(2)mbn cells were treated with ethanol (−) or ecdysone (+) for 30 min, and cross-linked chromatin was immunoprecipitated with EcR-B1 antibody, flag antibody for CARMER, and histone H3R17me2–specific antibody. CARMER was also knocked down by RNAi, and H3R17me2 was assessed. Input control represents 20% of genomic DNA from each treatment taken before the immunoprecipitation. For the detection of flag-CARMER, cells were transfected as outlined in Materials and methods with pIE CARMER. (B) Cells were treated as in A, but ecdysone treatment was performed for 10, 30, and 60 min. The indicated methylation-specific antibodies, dLKR/SDH antibody, and Ig control antibody (preimmune serum) were used for ChIP. rp49 was amplified as a control for the dLKR/SDH immunoprecipitation. Input DNA is shown (genomic). (C) EMSA was performed by ³²P labeling an oligonucleotide spanning the potential EcR/Usp BE of the ark promoter and incubating with 9 μg of nuclear extract and 1 μg EcR antibody that recognizes all isoforms (common), B1 isoform, α isoform, and control antibody N27A1 (N). Complexes were resolved on a 5% Tris/borate/EDTA acrylamide gel, dried, and exposed to a screen. ss, antibody super shift. (D) Genomic DNA samples from the same immunoprecipitations used in B were used to amplify regions of the ark promoter spanning the EcR/Usp BE. For the detection of flag-CARMER and dLKR/SDH, 2 × 10⁷ cells were transfected for each time point. (E) Schematic diagrams of dronc and ark promoters showing the locations of primers (arrows) used for ChIP analyses.