Manes G , Cheguru P , Majumder A , Bocquet B , Sénéchal A , Artemyev NO , Hamel CP , Brabet P .

EY-10843 NEI NIH HHS , EY-12682 NEI NIH HHS , R01 EY010843 NEI NIH HHS , R01 EY012682 NEI NIH HHS

	Figure 2. Clinical investigations of the index patient (III:1) of a French family with adCSNB.A. Fundus photographs of index patient III:1, right eye and left eye. B. Dark-adaptation curve of index patient III:1 (solid line) compared to normal curve (hatched line). D. Full-field ERGs from a normal control and index patient III:1.
	Figure 3. Interaction of PDE6β1-313 with the polycationic region of Pγ, Pγ24-45.A. Emission spectra of CysBC-Pγ24-45 (50 nM) alone (1) and in the presence of PDE6β1-313 (180 nM) (2) were recorded with with excitation at 445 nm. Inset: Coomassie Blue-stained gel with the sample of PDE6β1-313. B. Fluorescence of CysBC-Pγ24-45 (50 nM) was measured on addition of increasing concentrations of PDE6β1-313 (excitation at 445 nm, emission at 490 nm). The data were fit to the hyperbola equation using GraphPad Prizm software. C. Noncatalytic cGMP binding to PDE6β1-313. Binding of cGMP was carried out using [H3]cGMP and varying concentrations of unlabeled cGMP. The bound cGMP was determined by the filter-binding assay, and is expressed as a percentage of maximal cGMP binding (0.5 mol cGMP/mol PDE6β1-313). Results from one of three similar experiments are shown. The data were fit to equation for binding with ligand depletion using GraphPad Prizm software. The calculated Kd = 0.82±0.15 µM.
	Figure 4. Effect of PDE6β1-313 on the inhibition of trypsin-activated PDE6 (tPDE6) by Pγ.The activity of tPDE6 was determined upon addition of increasing concentrations of Pγ in the absence (squares) or in the presence (circles) of 2 µM PDE6β1-313. The tPDE6 activity is plotted as a function of Pγ concentration. Results from one of three similar experiments are shown. The data were fit to sigmoidal dose-response equation using GraphPad Prizm Software. The calculated IC50 values (squares, 0.53±0.07 nM; circles, 1.6±0.2 nM) are significantly different (p<0.01).
	Figure 5. Expression of PDE6β1-314fs*50 in transgenic X. laevis rods photoreceptors.A. left - EGFP fluorescence in living photoreceptor cells expressing PDE6β1-314fs*50, center - DIC image, right - EGFP-fluorescence/DIC overlay. Bar - 10 µm. B. Co-localization of PDE6β1-314fs*50 (green) with the rod outer segment marker Wheat Germ Agglutinin (red) in cryosections of transgenic retina. Cryosections were counterstained with TO-PRO3 nuclear stain (blue). Bar - 10 µm. C. Isotonic (I), hypotonic (H) extracts of transgenic PDE6β1-314fs*50 retinas were analyzed by immunoblotting with anti-GFP B-2 monoclonal antibody.
	Figure 6. Expression of EGFP-PDE6β1-313 in transgenic X. laevis rods photoreceptors.A. left - EGFP fluorescence in living photoreceptor cells expressing EGFP-PDE6β1-313, center - DIC image, right - EGFP-fluorescence/DIC overlay. Bar - 10 µm. B. Co-localization of EGFP-PDE6β1-313 (green) with the rod outer segment marker Wheat Germ Agglutinin (red) in cryosections of transgenic retina. Cryosections were counterstained with TO-PRO3 nuclear stain (blue). Bar - 10 µm. C. Isotonic (I), hypotonic (H) extracts of transgenic EGFP-PDE6β1-313 retinas were analyzed by immunoblotting with anti-GFP B-2 monoclonal antibody.
	Figure 1. Pedigree with adCSNB and PDE6B mutation.A. The three-generation French family MTP1481 with adCSNB. Filled symbols indicate affected family members; squares: males; circles: females; arrow: index patient; slashed symbols: deceased persons; question marks: family members with no information about the phenotype available. M indicates the presence of the mutation and + indicate the wild-type allele. B. Electropherograms show the normal control sequence and affected sequence (index individual III:1) surrounding the p.Tyr314Cysfs*50 mutation. The dotted line represents the 22 duplicated nucleotides. C. Schematic representation of the PDE6β protein showing the domain structure and positions of arRP truncating mutations (below) and adCSNB mutations (above) including the mutation described in this study (Y314Cfs*50).

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