Figure 3

Detection of Alexa Fluor 488-labeled P60 and P78 in rat vitreous after corneal application. (A) Fluorimetric detection of peptide levels in the vitreous. Peak levels of 0.2 ng/mL for P60 and 0.9 ng/mL for P78 were calculated at 0.5 and 4 h, respectively. Ratios of the peptide levels are given at the top of the graph bars (n = 15). Values are presented as means ± SEM. (B) Western blot (4–20% gradient SDS-PAGE) using an N-terminal PEDF polyclonal antibody confirms the presence of P60 and P78 in vitreous samples (20 µL) at 0.5 and 4 h, respectively, after the eye drops were given. Endogenous PEDF was also detected in the vitreous as the usual doublet migrating at ~50 kDa (M, molecular markers). (C) Densitometric analyses of Western blots indicate the difference in levels of the P60 and P78 peptides reaching the vitreous at the time points when their highest levels are seen by fluorometric analysis (A).