Muradov H , Boyd KK , Haeri M , Kerov V , Knox BE , Artemyev NO .

EY-10843 NEI NIH HHS , R01 EY010843-17 NEI NIH HHS , R01 EY010843 NEI NIH HHS

	FIGURE 1. Expression of EGFP-PDE6C in transgenic rods. A, map of the transgene. The XhoI site was used to linearize the pXOP(508/ﰂ41)EGFP-PDE6C plasmid for production of transgenic X. laevis embryos. B, EGFP fluorescence in living photoreceptor cells expressing EGFP-PDE6C and EGFP-Gﰆt. C, immuno- blotting of retinal extracts from transgenic (lanes 1 and 3) and non-transgenic retinas (lane 2) with anti-GFP B-2 monoclonal antibody (Santa Cruz Biotechnol- ogy, Inc., Santa Cruz, CA) (lanes 1 and 2) and anti-PDE6 MOE antibodies (CytoSignal) (lane 3). This research was originally published in The Journal of Biological Chemistry. Muradov et al “Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods.” The Journal of Biological Chemistry, November 20, 2009; 284 (47): 32662-32669. © The American Society for Biochemistry and Molecular Biology. Reproduced with permission.
	FIGURE 2. Localization of EGFP-PDE6C in transgenic rods. A and B, EGFP fluorescence in EGFP-PDE6C rod. A, Z-scans with an interval of 0.9 ﰈm through ROS of a light-adapted transgenic tadpole. Bar, 5 ﰈm. B, cross- section of the ROS in A corresponding to the position identified by arrows. Bar, 5 ﰈm. C, co-localization of EGFP-PDE6C with peripherin-2 in transgenic rods. Red, immunofluorescence of a retinal cryosection of a light- adapted transgenic tadpole stained with monoclonal antibody Xper5A11 against anti-Xenopus peripherin-2; green, EGFP fluorescence; overlay. This research was originally published in The Journal of Biological Chemistry. Muradov et al “Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods.” The Journal of Biological Chemistry, November 20, 2009; 284 (47): 32662-32669. © The American Society for Biochemistry and Molecular Biology. Reproduced with permission.
	FIGURE 3. Co-localization of EGFP-PDE6C with endogenous PDE6 in transgenic rods. A, red, immunofluo- rescence of a retinal cryosection of a light-adapted transgenic tadpole stained with anti-PDE6 MOE antibody; green, EGFP fluorescence; overlay. Bar, 10 um. B, MOE antibody immunofluorescence. Z-Scans with an interval of 1 um through the retina cryosection in A showing the rod cell identified by the arrow. Bar, 10 um. This research was originally published in The Journal of Biological Chemistry. Muradov et al “Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods.” The Journal of Biological Chemistry, November 20, 2009; 284 (47): 32662-32669. © The American Society for Biochemistry and Molecular Biology. Reproduced with permission.
	FIGURE 4. Analysis of lateral diffusion of EGFP-PDE6C by FRAP. A, photobleaching geometry to monitor lateral diffusion. The arrows indicate the direction of diffusion into the bleached stripe region. B, pre- and postbleach images of a transgenic ROS at different time points. The retina was incubated in Ringer buffer containing 6 mM 2-deoxyglucose and 10 mM NaN3. 150 images (256 ﰃ 256 pixels) were collected with a 1-s interval between the scans. C, intensity integrated along the y axis for each x axis pixel of the selected box region from the first postbleach image. The plot is used to determine the width of a bleach region with a Gaussian profile and the depth of bleach (21). D, intensities of the bleached region during the experimental time course corrected for background and fading and the fitting curve to a one-dimensional diffusion equation described previously (21). This research was originally published in The Journal of Biological Chemistry. Muradov et al “Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods.” The Journal of Biological Chemistry, November 20, 2009; 284 (47): 32662-32669. © The American Society for Biochemistry and Molecular Biology. Reproduced with permission.
	FIGURE 5. Analysis of longitudinal diffusion of EGFP-PDE6C by FRAP. A, photobleaching geometry to monitor longitudinal diffusion. The arrows indicate the direction of diffusion into the bleached stripe region. B, bleaches were introduced into two neighboring rods. Pre- and postbleach images of transgenic ROS at different time points. The retina was incubated in Ringer buffer containing 6 mM 2-deoxyglucose and 10 mM NaN3. 150 images (256 ﰃ 256 pixels) were collected with a 2-s interval between the scans. C, intensities of the bleached region from the left rod during the experimental time course corrected for background and fading and the fitting curve to a one-dimensional diffusion equation described previ- ously (21). This research was originally published in The Journal of Biological Chemistry. Muradov et al “Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods.” The Journal of Biological Chemistry, November 20, 2009; 284 (47): 32662-32669. © The American Society for Biochemistry and Molecular Biology. Reproduced with permission.
	FIGURE 6. Immunoprecipitation and solubilization of PDE6C. A, immunoblotting (WB) with anti-GFP B-2 monoclonal antibody (Santa Cruz Biotechnology, Inc.). Lane 1, retinal extract (0.5 transgenic retina equivalent); lane 2, control immunoprecipitation with unrelated rabbit anti-GST antibody; lane 3, proteins immunoprecipitated from retinal extract (four transgenic retinas) with anti-GFP antibody (ab290, Abcam). Lanes 4 and 5, EGFP standards: 5 and 15 ng of purified E. coli expressed EGFP-PDE6C (446 819). B, immunoblot with anti-PDE6 MOE antibody. Lane 1, transgenic retina extract; lane 2, proteins immunoprecipitated from retinal extract with anti-GFP antibody. C, PDE6 activity of the immunoprecipitated proteins from non-transgenic (left bars) and transgenic (right bars) extracts prior to and following trypsin treatment was measured using 200 ﰈM [3H]cGMP. D, after immunoprecipitation of EGFP-PDE6C with anti-GFP-antibody, the beads were treated with trypsin for 15 min. Proteins released into the soluble fraction were separated from the beads, reconstituted with soybean trypsin inhibitor, and analyzed by immunoblotting with anti-PDE6 MOE antibody. This research was originally published in The Journal of Biological Chemistry. Muradov et al “Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods.” The Journal of Biological Chemistry, November 20, 2009; 284 (47): 32662-32669. © The American Society for Biochemistry and Molecular Biology. Reproduced with permission.

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