Fig. 4.
Association of AaE74B with AaEcR and AaUSP. (A) Proteins were synthesized and were labeled by using the TnT Coupled Transcription/Translation System. These proteins were then incubated for 4 h in the presence or absence of 1 × 10-6 M 20E, followed by immunoprecipitation with the indicated antibodies. The resulting immune complexes were precipitated by addition of protein A-agarose beads. After extensive washing, the complexes were analyzed by SDS/PAGE and autoradiography. (B) Interaction of AaE74B with AaEcR and AaUSP in mammalian two-hybrid assay. CV-1 cells were cotransfected with control reporter pCMV-Luc and reporter construct pFR-Luc together with indicated plasmids. Luciferase activity was normalized with Renilla luciferase activity. The results represent the mean values obtained from three independent experiments. (C) Drosophila S2 cells were cotransfected collectively by vectors for AaE74B, AaEcRb, and AaUSPb and cultured in the presence or absence of 20E. Nuclear extracts prepared from the transfected cells were incubated with [35S]methionine-labeled AaEcRb, AaUSPb, or AaE74B, followed by immunoprecipitation.