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Subcellular localization of Npy2r and BAC-Npy2r-Cre transgenic mouse.

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posted on 08.06.2015, 03:08 by Yoshihiro Omori, Taro Chaya, Satoyo Yoshida, Shoichi Irie, Toshinori Tsujii, Takahisa Furukawa

A) The subcellular localization of Npy2r. FLAG-tagged Npy2r was expressed in the NIH3T3 cells. Cells were stained with the anti-acetylated α-tubulin (green) and anti-FLAG (red) antibodies. FLAG-tagged Npy2r signals were observed in NIH3T3 cells (red). Co-localization of Npy2r and acetylated α-tubulin signals was observed in cilia (arrowheads). Nuclei were stained with DAPI (blue). B) Diagram representing the BAC-Npy2r-Cre transgene construct. C–L) Expression of mTFP1 in the brains of R26-CAG-LoxP-mTFP1; BAC-Npy2r-Cre transgenic mice (C, D, G-I, K, L) and control mice (E, F). BAC-Npy2r-Cre transgenic mice were crossed with reporter mice, R26-CAG-LoxP-mTFP1, to detect the expression of the Cre recombinase in BAC-Npy2r-Cre transgenic mice. Dorsal (C, E), ventral (D, F, G) and coronal (H, I) views of the brain are shown. Broad expression of mTFP1 in the brain including the hippocampus, cerebral cortex, and hypothalamus (paraventricular nucleus, arrowheads in G, I) was observed. In the arcuate nucleus, Npy2r (green in J, arrowheads) colocalized with Adcy3 (a ciliary marker, red in J) in cilia. Npy2r-stained cilia (red) were often observed in mTFP1-positive cells (green in K). Preabsorption of the anti-Npy2r antibody with a synthetic Npy2r-peptide antigen abolished the Npy2r staining in the cilia (L). Scale bars, 10 μm (A, J), 5 μm (the inset in A), 5 mm (C–F), 1 mm (G–I), and 20 μm (K, L).