Fig. 8.

Role of PKA for α-MSH-induced dephosphorylation of AMPK at Thr¹⁷². Approximately 400,000 GT1-7 cells were grown on six-well plates, serum starved for 20 h, preincubated or not with (A) 100 ng/ml PTX over night, (B) with 10 μm gallein, (C) with 20 μm LY294002, (D) with 1 μm Gö6983 or BIM-X, (E) with 50 μm Rp-cAMPs, or (F) 5 μm KT5720 for 30 min, and stimulated with 1 μm α-MSH for 10 min. Lysates were then analyzed by Western blotting using a phospho-specific antibody against Thr¹⁷² of AMPK. In all cases, except from Rp-cAMPs, control cells were incubated with the carrier [0.1–0.5% dimethylsulfoxide (DMSO)]. One representative blot is shown. The dotted line indicates grouping of different lanes from the same gel. G, Data of seven independent experiments were compiled, normalized by setting values of unstimulated cells, DMSO, 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) differences between Rp-cAMPs-treated and basal cells or between KT5720 and DMSO (0.5%)-treated cells.