CFH SCR7 baits bind Fib3 prey in yeast 2-hybrid (Y2H) assays.

<p><b>a)</b> Cartoon of the Fib3 prey clone, corresponding to amino acid residues 289–493. EGF-like and C-terminal (CT) domains are indicated. Bars indicate the Fib3-EGF and Fib3-CT domains use to localize the CFH interaction site. <b>b)</b> Tabulation of relative colony growth on selective medium (4DO) for bait/prey combinations.</p

ELISA for recombinant constructs for CFH and Fib3 shows higher affinity binding for the CFH-H variant.

<p>a) Recombinant CFH678 constructs. Left: Coomassie stain for H and Y variants. Right: Western blot of constructs using goat anti-human CFH antibody. b) ELISA. Anti-HA antibody was anchored to the plate and used to capture HAFib3. Concentration ranges of CFH678 H and Y variants were added and detected with anti-CFH antibody, visualized with fluorescent labeled secondary antibody at measured at 450 nm. Measurements were replicated 8-fold and background for each point (ELISA with no added HAFib3) was subtracted. Curves were calculated by non-linear regression. Standard error of mean (SEM) is indicated for each point. The Scatchard plot for the same data is shown as an insert. Kd: CFH-H = 134 nM +/−16); CFH-Y: = 197 nM +/−29. Bmax: CFH-H = 0.8+/−0.03; CFH-Y = 0.7+/−0.03 (in OD units).</p

AMD and druse-containing eyes described in this paper.

<p>GA: geographic atrophy. All eyes were from white donors.</p

Patterns of Localization of CFH, Fib3 and cholesterol in AMD eyes vary with CFH genotype.

<p>Ph: photoreceptors; RPE: retinal pigment epithelium; BM: Bruch’s membrane. Scale bars: 50µM. <b>A:</b> Section of retina from (H/H) AMD donor #57985 labeled for CFH (red), Fib3 (rabbit polyclonal) (green), unesterified cholesterol (filipin, blue) and DAPI (white). Large soft drusen contain globules co-labeled for CFH and Fib3 embedded in cholesterol rich material. <b>B:</b> Section of retina from (H/H) AMD donor #68536 labeled as in A. This large druse also shows colocalization of CFH and Fib3 in a filipin positive region. Note that the relative intensities of CFH/Fib3 vary among deposits, leading to a range of merged colors. <b>C:</b> A region of macula from (Y/Y) donor #68280 with advanced wet AMD and fibrosis. There is a basal linear deposit strongly positive for Fib3 but lacking the globular structure and colocalization with CFH seen in H/H eyes. <b>D:</b> Section of eye from H/Y donor #68574 (wet AMD). No colocalization of CFH and Fib3. A small druse labeled with filipin (blue) is present (arrow). <b>E,F</b>: Foveal sections from H/Y “normal” donor #68259. <b>E:</b> Shows cones labeled with PNA (blue), rods labeled for rhodopsin (green), RPE labeled for RPE65 (red) and nuclei labeled with DAPI (white). Photoreceptors appear to be intact but overlie a soft druse (arrow). <b>F</b>: shows a region within the foveal druse showing punctate labeling for CFH (red) and Fib3 (green) in small adjacent deposits along Bruch’s membrane (arrow). Autofluorescence from lipofuscin granules in RPE is shown in magenta.</p

Validated prey clones from Y2H screen with CFH SCR7 domain baits.

<p>Compartment, function and domain information are taken from Entrez Gene <a href="http://www.ncbi.nlm.nih.gov/gene/" target="_blank">http://www.ncbi.nlm.nih.gov/gene/</a>.</p

CFH SCR7 402H variant binds Fib3 with higher affinity than the Y variant in Y2H.

<p>Yeast clones containing the Fib3 prey construct and either the CFH 402H (CFH7H) or 402Y (CFH7Y) bait constructs were tested in quantitative β-galactosidase assay. Controls shown are CFH7H bait with empty prey vector (pGAD) and Fib3 prey with empty bait vector (pGBTK7). Activity is expressed in Miller Units: ( = 1000×OD₅₇₈/t×V×OD₆₀₀, where t = incubation time in min; V = 0.1 concentration factor). Standard deviation error bars (N = 8) are shown.</p

Interaction of native CFH and Fib3.

<p><b>a) Fib3 expression is induced in serum-starved ARPE-19 cells in culture.</b> Western blot of serum-starved conditioned medium with anti-Fib3 antibody. For ARPE-19 cells, Fib3 is induced after 5 days in culture. No Fib3 is detectable in HEK293 cell conditioned medium after 7 days under the same conditions (H). <b>b) Co-immunoprecipitation of native CFH in presence of Fib3.</b> ARPE-19 (ARPE) or HEK 293 (HEK) cells were serum deprived for 5 days followed by addition of 0, 2 or 5 µg/ml of purified human Complement Factor. After 48 h conditioned media from each sample were subjected to immunoprecipitation with anti-Fib-3.ARPE and HEK lanes on the left show Western blot of media before IP using anti-CFH. Lanes on the right show western blots after immunoprecipitation. Co-IP of CFH with Fib3 antibody occurs only in ARPE-19 medium which contains Fib3 (as shown in Fig. 2a). c) <b>Co-immunoprecipitation of native Fib3 in presence of CFH.</b> Medium from 7 day serum starved ARPE-19 cells with or without addition of 2µg human CFH was immunoprecipitated with antibody to CFH and Fib3 was detected by Western blot.</p

Immunofluorescence of CFH, Fib3 and controls in normal and dry AMD eye GCL (ganglion cell layer), INL (inner nuclear layer), ONL (outer nuclear layer), RPE (retinal pigment epithelium), CH (choroid).

<p>Nuclei (gray) and autofluorescence from lipofuscin granules (magenta) are visible in all cryosections. Scale bar = 50µM. <b>A:</b> Section of retina from a normal human eye labeled with mouse monoclonal antibody against Fib3, mFib3 (green) and an anti-CFH antibody (red). In normal retina, the Fib3 immunoreactivity is present in the ganglion cell layer as reported previously. CFH is restricted to choroidal blood vessels. <b>B</b>: Section from H/H AMD eye (#57985) with soft druse. CFH (red) is detected in globules within the druse. <b>C:</b> Similar section to B labeled for both mFib3 (green) and an anti-CFH antibody (red). Strong Fib3 immunoreactivity is observed in the soft druse (arrows) and the signal colocalizes with that of anti-CFH. Merged colors vary according to relative staining for both antibodies. <b>D:</b> The colocalization of Fib3 and CFH in soft drusen (arrows) is confirmed with a newly generated anti-fib3 antibody-rFib3 (rabbit polyclonal antibody raised against the N-terminal region of human Fib3 (blue). The two independently derived anti-Fib3 antibodies show identical staining patterns. (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068088#pone.0068088.s003" target="_blank">Fig. S3</a>). <b>E:</b> Little labeling of either Fib3 or CFH was observed in a hard druse (*) from the same AMD donor (left panel). <b>F:</b> Control antibodies for markers for retinal glia (anti-GFAP rabbit polyclonal, blue) and rod photoreceptors (anti-rhodopsin mouse monoclonal, green) do not label CFH-positive areas within the soft drusen (arrows). <b>G–I:</b> Detailed region of a similar soft druse from H/H AMD donor #68536. <b>G:</b> green label for Fib3; <b>H:</b> red label for CFH; <b>I:</b> merged image. Arrows point out some of the deposits showing similar levels of labeling for both antibodies. Scale bar = 50µM.</p

Part of a candidate disease region (AXPC1) showing a novel spliced gene transcript

This view shows a detail of one end of the AXPC1 region. An expressed sequence tag (EST) from the retinal pigment epithelium (RPE) shows the structure (vertical bars are exons; arrowed lines show introns) of a novel gene. EyeSAGE has tag counts for some Unigenes in this region, including the novel gene (designated as C1orf132 in Unigene). Note that the EyeSAGE tag bars are positioned according to the corresponding Unigene. Their positions may change slightly with different releases of Unigene.<p><b>Copyright information:</b></p><p>Taken from "NEIBank: Genomics and bioinformatics resources for vision research"</p><p></p><p>Molecular Vision 2008;14():1327-1337.</p><p>Published online 18 Jul 2008</p><p>PMCID:PMC2480482.</p><p></p