(A) qRT-PCR analysis of drPanx1 expression. Total RNA was isolated from several CNS regions of adult zebrafish und used for cDNA synthesis. Retinal drPanx1 expression was normalized to 1. Each bar represents the mean + SEM. (n = 3) (B-D) IHC/DAB staining of drPanx1b expression in the retina. The anti-drPanx1b antibody was used as primary antibody. Cell bodies were stained with propidium iodide (red; B) or cresyl violet (violet; C, D). Arrows point at drPanx1b positive cells in the INL and GCL. (B) The primary antibody was visualized by an anti-chicken antibody conjugated with Alexa488. (C, D) For DAB staining, a biotinylated anti-chicken antibody served as detection antibody. (D) Control staining using the immunizing blocking peptide together with the anti-drPanx1b antibody. (Scale bar (B-D) = 10 µm; GCL = ganglion cell layer; INL = inner nuclear layer; IPL = inner plexiform layer; ONL = outer nuclear layer; OPL = outer plexiform layer; PR = photoreceptors).

(A) Localization of Panx1-EYFP WT and glycosylation-deficient drPanx1b-EYFP mutant proteins in N2a cells. Protein-derived fluorescence was detected using confocal laser scanning microscopy 48 h post transfection. (Scale bar = 10 µm) (B) Western blot analyses of Panx1-EYFP WT and (C) glycosylation-deficient drPanx1b-EYFP mutants. N2a cells transiently transfected with pEYFP-N1 expression vector constructs were lysed 48 h post transfection. 20 µg total protein were subjected to SDS-PAGE and subsequent western blot analyses using the mouse anti-GFP and anti-mouse IRDye680RD antibodies (B) or rabbit anti-GFP and anti-rabbit IRDye680LT for detection of the Panx1 fusion proteins (C). Mouse anti-β-actin served as a control for equal protein loading and was detected using anti-mouse IRDye680RD (B) or anti-mouse IRDye800CW (C). Arrowheads in (C) indicate the six drPanx1b bands.