The steroid hormone 20-hydroxyecdysone binds to dopamine receptor to repress lepidopteran insect feeding and promote pupation
Fig 10
A. Saturation binding curves of DopEcR-GFP and DopEcR-M-GFP to 20E. B. Saturation binding curves of ErGPCR-2-GFP and ErGPCR-2-M-GFP to 20E. Nonspecific binding was analyzed with GFP-His tag. All the experiments were performed using 10 μg of isolated protein in 50 μL EIA buffer. C. SDS-PAGE to show the highly purified GPCRs used in A, B, F and G. D. The cAMP levels of HaEpi cells overexpressed with DopEcR-GFP, DopEcR-M-GFP, ErGPCR-2-GFP, and ErGPCR-2-M-GFP under 20E-triggered. HaEpi cells were transfected with DopEcR-GFP, DopEcR-M-GFP, ErGPCR-2-GFP, and ErGPCR-2-M-GFP for 48 h followed by incubation with 2 μM 20E. E. Ca2+ levels after DopEcR-GFP, DopEcR-M-GFP, ErGPCR-2-GFP, and ErGPCR-2-M-GFP overexpressed in HaEpi cells. Cells were transfected with DopEcR-GFP, DopEcR-M-GFP, ErGPCR-2-GFP, and ErGPCR-2-M-GFP for 48 h and AM ester calcium crimson dye (3 μM) in DPBS for 30 min, and then by 20E (1 μM) and CaCl2 (1 mM), respectively. F: fluorescence intensity of HaEpi cells after different treatments. F0: fluorescence intensity before different treatments. Fluorescence was recorded per 6 s by confocal microscope photographs at 555 nm wavelength laser and then analyzed using Image Pro-Plus software. F. Saturation binding curves of DopEcR-GFP to DA. Nonspecific binding was analyzed with GFP-His tag. G. The competition curves of DopEcR-GFP to 20E and Dopamine. The ELISA plate coated with DopEcR-GFP was incubated with 20 pmol of DA in the presence of increasing concentrations of the different ligands (20E and DA). Error bars represent the SD of three replicates. Asterisks indicate significant differences according to Student’s t-tests (*p < 0.05; **p < 0.01).