Specimens and samples used for immunohistrochemistry and western blotting analysis.

<p>Specimens and samples used for immunohistrochemistry and western blotting analysis.</p

OXA and OXB single immunohistochemical detection.

<p>OXA immunopositive perivascular fibers in the submucosal layer of main stomach (a); OXA ir neurons and nervous fibers in the myenteric plexus of proximal intestine (b); OXB ir neurons of the submucosal plexus of pyloric stomach (c) and myenteric plexus of proximal intestine (d). Scale bars: 5 µm (a); 20 µm (b); 10 µm (c, d).</p

Orexin system protein expression.

<p>Western blot for orexin 1 and 2 receptors, prepro-orexin and β-actin (used as internal marker) in forestomach, main stomach, pyloric stomach, proximal and distal intestine.</p

OXA plasma levels.

<p>Bottlenose dolphin plasma OXA concentration (mean ± SD) at 10∶00 and 17∶30 hours.</p

Specimens and samples used for immunohistrochemistry and western blotting analysis.

<p>Specimens and samples used for immunohistrochemistry and western blotting analysis.</p

Data_Sheet_2_Overlapping Distribution of Orexin and Endocannabinoid Receptors and Their Functional Interaction in the Brain of Adult Zebrafish.docx

<p>Hypocretins/Orexins neuropeptides are known to regulate numerous physiological functions, such as energy homeostasis, food intake, sleep/wake cycle, arousal and wakefulness, in vertebrates. Previous studies on mice have revealed an intriguing orexins/endocannabinoids (ECs) signaling interaction at both structural and functional levels, with OX-A behaving as a strong enhancer of 2-arachydonoyl-glycerol (2-AG) biosynthesis. In this study, we describe, for the first time in the brain of zebrafish, the anatomical distribution and co-expression of orexin (OX-2R) and endocannabinoid (CB1R) receptors, suggesting a functional interaction. The immunohistochemical colocalization of these receptors by confocal imaging in the dorsal and ventral telencephalon, suprachiasmatic nucleus (SC), thalamus, hypothalamus, preoptic area (PO) and cerebellum, is reported. Moreover, biochemical quantification of 2-AG levels by LC-MS supports the occurrence of OX-A-induced 2-AG biosynthesis in the zebrafish brain after 3 h of OX-A intraperitoneal (i.p.; 3 pmol/g) or intracerebroventricular (i.c.v.; 0.3 pmol/g) injection. This effect is likely mediated by OX-2R as it is counteracted by i.p./i.c.v administration of OX-2R antagonist (SB334867, 10 pmol/g). This study provides compelling morphological and functional evidence of an OX-2R/CB1R signaling interaction in the brain of adult zebrafish, suggesting the use of this well-established vertebrate animal model for the study of complex and phylogenetically conserved physiological functions.</p

zBcl10 activates NF-κB.

<p>A) HEK293 cells were transiently cotransfected with an expression vector empy (<i>vector</i>) or encoding for the indicated polypeptides, together with pNF-κB-luc and pRSV-βgal reporter vectors. The total amount of transfected plasmidic DNA was maintained constant by adding empty vector. 16 hrs after transfection, cell lysates were prepared and luciferase activity was measured. In the bottom panels, a fraction of the cell lysates were analyzed by immunoblot to monitor protein expression. Data shown (mean + SEM, n = 9) represent relative luciferase activity normalized on β-galactosidase activity and is representative of six independent experiments done in triplicate. Statistical analysis was performed by Student's t test; a p value of <0.05 was considered significant, and is indicated with the symbol *. B) HEK293 cells were transiently cotransfected with an expression vector empy (<i>vector</i>) or encoding zebrafish and human BCL10 with or without the de-ubiquitinase A20. 16 hrs after transfection, cell lysates were prepared and luciferase activity was determined as in <i>A)</i>.</p

zBcl10 dimerizes and binds to CBM proteins.

<p>A) HEK293 cells were transiently cotransfected with FLAG-tagged and HA-tagged version of zBcl10 and hBCL10 or empty vector (<i>vector</i>). 24 hrs later, cell lysates were immunoprecipitated with anti-FLAG mAb. Immunocomplexes were separated by SDS-PAGE and transferred onto membranes subsequently probed with anti-HA antisera. <i>B-C)</i> Lysates from HEK293 cells transfected with HA-zBcl10 were immunoprecipitated with anti-MALT1 (B) or anti-CARMA3 (C) and analyzed for coprecipitating HA-zBcl10. D) HEK293 cells were transiently cotransfected with HA-zBcl10 and FLAG-tagged CARMA2<i>sh</i> or empty vector. Lysates were immunoprecipitated with anti-FLAG mAb and analyzed for coprecipitating HA-zBcl10 by immunoblot assay.</p

zBcl10 activates NF-κB in zebrafish cells.

<p>A) PAC2 cells were transiently cotransfected with expression vectors encoding for the indicated polypeptides, together with pNF-κB-luc and pRSV-βgal reporter vectors. The total amount of transfected plasmidic DNA was maintained constant by adding empty vector. 24 hrs after transfection, cell lysates were prepared and luciferase activity was measured. Data shown (mean + SEM, n = 9) represent relative luciferase activity normalized on β-galactosidase activity and is representative of six independent experiments done in triplicate. Statistical analysis was performed by Student's t test; a p value of <0.05 was considered significant, and is indicated with the symbol *. B, C) HEK293 and PAC2 cells were transiently cotransfected with expression vectors encoding for wt CARMA2<i>sh</i> or the psoriasis-linked mutants CARMA2<i>sh</i>E138A, together with pNF-κB-luc and pRSV-βgal reporter vectors and luciferase activity was determined as described in A).</p

Alignment and phylogenetic tree of zBcl10.

<p>A) Alignment of zBcl10 and human BCL10 with the consensus sequence generated by aligning the BCL10 sequences of fish, birds and reptiles, and mammals. The Xenopus tropicalis sequence is the only amphibian BCL10 sequence available. The alignment was done using ClustalW. Printout from multiple-aligned sequences and consensus sequences calculation were done with BOXSHADE. The black background designates identical amino acids, the gray background conservative substitutions. The red rectangles indicate amino acids conserved in all sequences examined. At the top of the alignment, the six alpha helix regions of the CARD are shown. The sequences used for alignment and generation of the consensus sequence are available in Supplementary Material. B) Phylogenetic tree analysis of BCL10 proteins. Phylogenetic analyses with bootstrapping (100 replicates) were obtained by the Neighbor-joining method using complete deletion and the p-distance amino acid model in MEGA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122365#pone.0122365.ref028" target="_blank">28</a>]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches. The sequences used for phylogenetic tree generation are available in Supporting Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122365#pone.0122365.s001" target="_blank">S1 Table</a>).</p