Figure 5
From: G-protein-coupled receptor participates in 20-hydroxyecdysone signaling on the plasma membrane
ErGPCR is located on the plasma membrane and is involved in 20E-induced gene expression in HaEpi cells. (A) Green fluorescence indicated ErGPCR protein detected with anti-ErGPCR (antibody specificity is shown in Additional file 1: Figure S7) and ALEXA 488–labeled goat anti-rabbit secondary antibody by confocal microscope. The nuclei were stained with 4′-6′-diamidino-2-phenylindole dihydrochloride (DAPI). (B) Green fluorescence protein (GFP, green) alone was detected in the entire cell; the plasma membrane (red) was marked with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI); and the orange color are the overlapping of ErGPCR-GFP (green) and membrane (red) by confocal microscope. Scale bar = 20 μm. (C) and (D) The effects of ErGPCR knockdown and overexpression on 20E-induced gene expression, analyzed by qRT-PCR. (E) and (F) 20E upregulated mod(mdg4)1a expression not through EcRB1 but through ErGPCR, by qRT-PCR analysis. β-actin was used as the quantitative control. * indicates significant differences (p < 0.05) among the treatments via Student’s t test from three independent replicates.