Figure 3. .

Increased VEGF levels in cbs^−/− mouse retinas. (A) Fluorescent immunodetection of VEGF (green), a marker of neovascularization, and isolectin-B4 (red), a marker of endothelial cells, was performed in retinal cryosections from 3-week cbs^+/+, cbs^+/−, and cbs^−/− mice. The far left panel provides controls: an isotype (IgM) control for VEGF and a negative control (omission of the primary antibody), but with inclusion of the secondary antibody, for isolectin-B4. Scale bar: 50 μm. Abbreviations for retinal layers are the same as for Figure 1. (B) Quantification of the intensity levels of VEGF immunofluorescence, showing significantly greater VEGF levels in cbs^−/− compared to cbs^+/+ and cbs^+/− mice (***P < 0.001, n = 6). (C) Expression of VEGF mRNA is increased in neural retinas of cbs^−/− mice compared to cbs^+/− and cbs^+/+ mice. Fold change in expression of vegf₁₂₀ and vegf₁₆₄ was determined by quantitative RT-PCR (*P < 0.05). (D) Representative immunoblot to detect VEGF in retinas of cbs^+/+ mice compared with cbs^+/− and cbs^−/− mice. Protein was extracted from retina and subjected to SDS-PAGE; immunoblotting was performed with an affinity-purified antibody against VEGF 164 and subsequently with an antibody against β-actin (internal loading control).