Fig. 5

miR-9a inhibits ac2 mRNA and protein expression by direct interaction. a Co-localization of miR-9a and ac2 in the locust brain through the co-labeling of miRNA and mRNA with fluorescence in situ hybridization. The squares specifically indicate the areas where miR-9a and ac2 were localized in the Kenyon cells of mushroom body. A white signal is observed in the area with magenta (miR-9a) and green signals (ac2) overlapping, indicating the co-localization of miR-9a and its target. The images were visualized using an LSM 710 confocal fluorescence microscope (Zeiss) at magnifications of ×10 (the upper row) and ×20 (the bottom row). Scale bar represents 20 μm. b RNA immunoprecipitation (RIP) was performed with an anti-Ago-1 antibody; normal mouse IgG was used as a negative control. RT-PCR or qPCR analysis was performed to amplify ac2 mRNA from the Ago-1 immunoprecipitates in brain tissue extracts treated with agomir-9a compared with agomir-control (agomir-NC). c, d Relative mRNA level (c, n = 6) and protein level (d, n = 6) of ac2 in the brains of the gregarious locusts injected with agomir-9a. e, f Relative mRNA level (e, n = 6) and protein levels (f, n = 6) of  ac2 in the brains of the solitarious locusts injected with antagomir-9a. The data for RIP assay, qPCR, and Western blot are presented as the mean ± SEM; The asterisks outside the strip indicate the significant difference between controls and the treatments by Student’s t-test. *P < 0.05; **P < 0.01. S: solitarious, G: gregarious, NC: negative control