Figure 2. The distribution of high-mobility group box protein 1 (HMGB1) in retinal ganglion cell (RGC)-5 cells changed following treatment with advanced glycation end products–BSA (AGE-BSA). A: Immunofluorescence photographs (400× magnification) show that HMGB1 appears in the cytoplasm (arrow) of RGC-5 cells at 3 h after treatment with 200 μg/ml of AGE-BSA, while HMGB1 remains in the nucleus of RGC-5 cells treated with either BSA (200 μg/ml) or PBS. The left column shows the distribution of HMGB1 protein with green fluorescence. The central column shows the nucleus stained with 4',6-diamidino-2-phenylindole (DAPI; blue), and the right column shows merged pictures. B: Subcellular fractionation of proteins shows a significant increase of HMGB1 levels in the cytosol of RGC-5 cells after incubation with AGE-BSA for 3 h (n=4, p<0.05, BSA-treated cells were used as control). The level of HMGB1 in the nucleus was not different between groups over time.