Figure 8. Western blot analysis of GSK-3β and GS phosphorylation and BAX, Bcl-2, pBcl-2, and phospho-c-Jun in secondary cultures of normal bovine cells treated with UO126 inhibitor. Bovine cell cultures were incubated for 90 min in serum-free minimal essential media (MEM), under conditions of atmospheric oxygen, containing either 10 µM UO126 or 0.05% DMSO) vehicle. Cells were then exposed to hypoxia for 3 h in the presence of either UO126 or DMSO vehicle. At the end of the hypoxic incubation period, the hypoxic media were removed, and fresh, oxygenated serum-free MEM with UO126 or DMSO 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. Prevention of phosphorylation of ERK with UO126 treatment was noted (top panel), as was the inhibition of phosphorylated glycogen synthase with western blot analysis (middle panel). The levels of GSK-3β) and pGSK-3β were consistent in the presence and absence of UO126. The phosphorylation of c-Jun, as well as that of Bcl-2, was blocked by treatment with UO126 under all conditions as determined with western blot analysis (bottom panel). Of particular note, Bcl-2 levels were unaffected by UO126 treatment compared to HLE-B3 cells (refer to Figure 5) where a significant diminution of Bcl-2 was observed but only upon reintroduction to atmospheric oxygen. No change in the relative levels of BAX was evident by treatment with UO126 under any condition. The experiment with normal, secondary cultures of bovine cells was run once, to confirm that a similar pattern of biochemical modifications by treatment with UO126 was reproducible with normal bovine cell cultures as was observed with HLE-B3, affirming that the former results were not influenced by viral transformation.