Figure 2. AGE-modification of FN alters integrin-mediated signaling in RMECs. A: western blotting analysis showed that phospho-FAK (tyrosine³⁹⁷) was reduced when RMECs were grown on AGE-FN. Quantification revealed this clear differential, which is especially evident at the 45 and 60 min time point. B-G: Confocal microscopy disclosed integrin α5β1 immunoreactivity in RMECs grown on FN and AGE-FN. B-D: RMECs cultured on native FN exhibited a relatively uniform distribution of α5β1 throughout the basal aspect of the cell immediately adjacent to the substrate. Deeper (basal) z-sections showed relatively weak fluorescence intensity at the basal plasma membrane (basal PM; D). E-G: RMECs cultured on AGE-FN exhibited a greater intensity of green fluorescence indicative of higher α5β1 expression. Some RMECs exposed to AGE-FN showed perinuclear punctuate staining (F). Deeper z-sections showed high fluorescence intensity and a filamentous distribution at the basal PM (G). All cells were counterstained with propidium iodide.