Presence of oxDJ-1 in RPE cells subjected to oxidative stress.

<p>ARPE-19 monolayers were treated with increasing concentrations (0 to 800 µM) of H₂O₂ for 1 hr (A) and 18 hs (B), harvested, and analyzed by immunoblot assay with oxDJ-1 antibody (upper panel). Protein loadings were confirmed in replicate blots probed with GAPDH (lower panel). Each lane contained 20 µg of protein. A dose response is observed when cells are exposed to increasing concentrations of H₂O₂ for 1 h (A, lanes 1 to 6) and 18 hrs (B, lanes 7 to 12). Confocal immunofluorescence staining of baseline ARPE-19 cultures (C) fixed before extraction with Triton X-100 and labeling with oxDJ-1 antibodies revealed absence of oxDJ-1. However, oxDJ-1 is observed in the cytoplasm (arrows) and perinuclear area (arrowheads) of RPE cells exposure to 400 µM H₂O₂ for 1 h (D) and 18 hrs (E). Cell nuclei were labeled with TO-PRO-3. Scale bar = 20 µm.