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. 2015 Jul 1;290(33):20438–20447. doi: 10.1074/jbc.M115.657379

FIGURE 2.

FIGURE 2.

DHA increases AMPK phosphorylation by releasing intracellular Ca2+. A, C2C12 cells were pretreated with fluo-3 AM for 30 min and then with 100 μm DHA. Green fluorescence was detected using confocal microscopy and analyzed. B, C2C12 cells were pretreated with STO-609 for 30 min and then with 50 μm DHA for 3 h. The cell lysates were analyzed by Western blotting with antibody against phospho-AMPK. AMPK and β-actin served as controls. The results are representative of four independent experiments. *, p < 0.05 versus DHA-treated conditions. C, L6 myoblasts were differentiated for 7 days and were pretreated with STO-609 (2 μm) and DHA (50 μm) for 1 h. Uptake of 2-DG was assayed. *, p < 0.05 compared with control. D, C2C12 cells transiently transfected with CaMKKβ siRNA for 2 days and stimulated using 50 μm DHA for 1 h. The cell lysates were analyzed by Western blotting with antibody against phospho-AMPK. AMPK and β-actin served as controls. The results are representative of four independent experiments. *, p < 0.05 versus DHA-treated conditions. E, L6 myoblasts were transiently transfected with CaMKKβ siRNA for 2 days and stimulated using 50 μm DHA for 1 h, and 2-DG uptake was assayed. *, p < 0.05 compared with control. IB, immunoblot.