Figure 3. Expression and activation of phospho-c-Jun and c-Jun protein in human scleral fibroblasts treated with 1 µmol/l all-trans retinoic acid. Panel A shows expression of phospho-c-Jun protein in HSFs visualized with indirect immunofluorescence. The nuclei were stained with propidium iodide dye (red: A1, B1, C1) and the primary antibody was labeled with daylight 488-conjugated secondary antibody (green: A2, B2, C2). A1-A3 shows a negative control incubated in 0.01 M phosphate buffered saline (PBS) with no primary antibody. B1-B3 shows cells incubated in control medium. C1-C3 shows upregulation of phospho-c-Jun protein expression in cells incubated with all-trans retinoic acid (ATRA) for 1 h. The original magnification was 400 X, and the scale bar=20 µm. Panel B shows western blot for phospho-c-Jun, c-Jun, and antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH). There is significant upregulation of phospho-c-Jun protein after incubation with 1 µmol/l ATRA from 10 min to 30 min, but no change of c-Jun protein. The bar graph in figure 3C shows significant and increasing upregulation of phospho-c-Jun protein expression relative to GAPDH density in cells incubated with ATRA from 10 min to 30 min (n=3, *p<0.05). In contrast, as seen in the bar graph of panel D, incubation with ATRA did not change expression of c-Jun protein relative to GAPDH density (n=3).