Fig. 6

Quantitative analysis revealed that truncated FH variants modulate C3 fragmentation in the ischemic retina. A Schematic representation of C3 cleavage. Numerical data indicate peptide sizes in kilodalton (kDa). The binding sites for complement receptors 1 and 2 (CR1 / CR2), FH, MCP and factor B (FB) are highlighted. C3 convertase cleaves C3 into C3b and C3a. With MCP, CR1 and FH as cofactors cleaves FI C3b into iC3b and C3f. Subsequent cleavage of iC3b by FI leads to C3c and C3dg, the latter being further converted to C3d. Modeled after [34]. B Representative Western blot of AAV treated retinas containing 70 µg total protein. Detection was performed by using a polyclonal antibody specific for C3d. (C3α + x corresponds to C3 α-chain covalently attached to glycoproteins via thioester group.) Original documentation of the blot is shown in Additional file 1: Fig. S5. C–H Quantitative analysis of Western blot band intensities normalized to total protein loading. Boxplots represent median, quartiles and mean (indicated by a dot) without outliers. The mean of each data set was compared with the AAV_EGFP ischemia data using Dunnett’s test for multiple comparisons. A significance level is indicated by *P < 0.05. Each data point per biological replicate (n = 3–4) is represented by a dot in the graph. Uncropped blots are summarized in Additional file 1: Fig. S6. I Immunolabeling with an antibody against C3d (labeling C3, C3b, iC3b, C3dg and C3d) showed co-staining with the Müller cell marker glutamine synthetase (GLUL) in the GCL of the ischemic retina. Application of AAV_FH1-4^19-20 reduced C3 intensity at 3 dpi compared with AAV_EGFP, whereas treatment with AAV_FH1-7^19-20 resulted in a reduction after 14 dpi. Each data point per biological replicate is represented by a dot in the graph. Unpaired t-test: *P < 0.05; **P < 0.01; ***P < 0.001