Figure 2. Western blot analysis of glycogen synthase kinase 3β and glycogen synthase phosphorylation in HLE-B3 cells in the presence or absence of SB216763. Total cell lysates were collected from >85% confluent human lens epithelial (HLE-B3) cell cultures that were incubated for 90 min in serum-free minimal essential media (MEM), under conditions of atmospheric oxygen, containing either 12 µM SB216763 or 0.05% DMSO vehicle. Cells were then exposed to hypoxia for 3 h in the continued presence of SB216763. At the end of the hypoxic incubation period, the hypoxic media were removed, and fresh, oxygenated serum-free MEM with SB216763 or DMSO vehicle were added to the cultures. Cells were then placed in atmospheric oxygen for up to 3 h. Cultures were collected after (1) continuous normoxic exposure (about 21% oxygen), (2) after the 3 h hypoxic exposure (about 1% oxygen), or (3) after reintroduction of atmospheric oxygen (about 21%) for 1, 2, or 3 h subsequent to the 3 h hypoxic exposure. Total cell lysates were analyzed with immunoblots using 25 µg of protein per lane. Anti-actin was used to normalize the bands to ensure equivalent lane loading. Three experiments, using independent cell populations, were quantified using GraphPad Prism 5 and the relative densities plotted for pGSK-3β/GSK-3β and pGS/GS. No change was evident in the ratio of pGSK-3β/GSK-3β while significant inhibition of the phosphorylation of glycogen synthase by SB216763 was noted. Error bars represent standard error. The asterisks (***) indicate p<0.001, Student t test.