Research Paper Volume 12, Issue 14 pp 13905—13923

Activation of C-reactive protein proinflammatory phenotype in the blood retinal barrier in vitro: implications for age-related macular degeneration

Figure 3. Diffusion of CRP isoforms across primary porcine RPE cells. (A) Western blot of mCRP present in apical (Up) and basolateral (Down) supernatants 48 hours after addition of mCRP (N=4). (B) ELISA of mCRP (ng/ml) from apical (Up) and basolateral (Down) supernatants. Values are expressed as mean ± SD (N=5). (C) Western blot of pCRP present in apical (Up) and basolateral (Down) supernatants 48 hours after treatment (N=3). (D) Immunofluorescence of CRP (red) stained with monoclonal antibodies against mCRP (3H12) or pCRP (1C6). Nuclei stained with DAPI. Scale bar = 30 μm (N=3). (E) Quantification of CRP binding measured as stained area divided by the number of cells per image (μm2/cell). Results are expressed as mean area (μm2/cell) ± SD. Statistical analysis was performed by One-Way ANOVA and Tukey’s posthoc. **P<0.01 vs. all conditions. (F) Reconstruction of x-z sections with a 0.3 μm z axis step of immunofluorescence images. Images shown are representative of three independent experiments.