Fig. 6.

Role of ERK-1/2 for basal and α-MSH-induced dephosphorylation of AMPK at Thr¹⁷². A, Approximately 400,000 GT1-7 cells were grown in six-well plates, serum starved for 20 h, and stimulated with either PD98059 (50 μm), U0126 (10 μm), or PD184352 (10 μm) for 20 min. Lysates were then analyzed by Western blotting using either phospho-specific antibodies against Thr¹⁷² of AMPK and against phosphorylated ERK-1/2 or against unmodified proteins (t-ERK-2 or t-AMPK). One representative blot for each antibody is shown. B, Phosphorylation of AMPK was monitored as described in A. Additionally, cells were preincubated with 10 μm U0126 or PD184352 or carrier [0.5% dimethylsulfoxide (DMSO)] for 20 min and then stimulated or not with 1 μm α-MSH for 10 min. One representative blot is shown. Data of seven independent experiments were compiled, normalized by setting values of unstimulated cells (basal) or inhibitor-treated cells as 100%. α-MSH-induced dephosphorylation was calculated by subtracting values of stimulated cells from 100% and is shown as the mean ± sem. Asterisks indicate a significant (***, P < 0.001) difference to zero. Hashed signs indicate a significant (##, P < 0.01) difference between with U0126 or PD184352-treated and DMSO-treated cells.