Fig. 1. Structure and expression of XNOS.a, Structure of XNOS cDNA. cDNA (6.5 kb) was cloned from a stage 42 Xenopus cDNA library. It corresponds to a protein of 1419 aa, which has all of the regions of neuronal NOS isoforms conserved between mammals and Drosophila.FAD, Flavin-adenine dinucleotide; FMN, flavin mononucleotide. b, Expression of XNOS. Western blots of protein extracts were probed using a rat nNOS- and XNOS-specific polyclonal antibody. Each lane was loaded with 25 μg of protein isolated from 293 cells transfected with rat nNOS cDNA (first lane), 293 cells transfected with XNOS cDNA (second lane), and adultXenopus frog brain (third lane). The blot was treated with R20 polyclonal antibody raised against a 20 aa peptide, which is identical in both rat nNOS (positions 1400–1419) andXenopus XNOS (positions 1390–1409). c, Enzymatic activity of recombinant XNOS. 293 cells were transfected with XNOS cDNA, and the NOS activity in the cell extract was measured using the arginine-to-citrulline conversion assay in the presence or absence of various cofactors and inhibitors. d, In situ hybridization of XNOS RNA probe in whole-mount stage 47 tadpole brain and eyes. ret, Retina;telen, telencephalon; tectum, optic tectum; mb, midbrain; hb, hindbrain;R, rostral; C, caudal. e, Whole-mount NADPH-diaphorase staining of the tadpole brain at stage 46. The region depicted corresponds to the central left portion of d. Light stainingillustrates NADPH-diaphorase-positive cell bodies in the caudal optic tectum. Note that their position is similar to that of XNOS-positive cells in d. The cluster at thebottom corresponds to the XNOS-positive cells of the hindbrain in d. The dotted line marks the optic tectum; M, midline. Scale bars: d, 100 μm; e, 25 μm.
Image published in: Peunova N et al. (2001)
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