Fig. 5.

Role of LKB-1 for α-MSH-induced dephosphorylation of AMPK at Thr¹⁷². Approximately 400,000 GT1-7 cells were grown in six-well plates, serum starved for 20 h, and analyzed by Western blotting using a phospho-specific antibody against Thr¹⁷² of AMPK. A, Cells were pretreated for 30 min with the CaMKKβ inhibitor STO609 (1 μm) or the TAK-1 inhibitor oxozeaenol (500 nm), respectively, and then stimulated or not with 1 μm α-MSH for 10 min. B, Serum-starved cells were incubated with the Hsp90 inhibitor radicicol (10 μm) for 20 h or as a control with the carrier ethyl alcohol (EtOH) (1%) and then stimulated or not with 1 μm α-MSH for 10 min. One representative blot is shown in each case. Data of five (A) or six (B) independent experiments were compiled, normalized by setting values of unstimulated cells (basal) or with 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; **, P < 0.01) difference to zero. Hashed signs indicate a significant (##, P < 0.01) difference between radicicol-treated and EtOH-treated cells. C, Cells were transfected with a siRNA designed to specifically down-regulate LKB-1 and a random siRNA (neg), serum starved for 20 h, and then stimulated or not with 1 μm α-MSH for 10 min. Samples were analyzed by Western blotting using an antibody either against AMPK-Thr¹⁷², LKB-1, p-ERK-1/2, ERK-2, or t-AMPK-α. Representative blots out of three independent experiments are shown. D, Data of three independent experiments were compiled, normalized by setting basal values of random siRNA (neg)-expressing cells or siRNA-1-expressing cells as 100%. α-MSH-induced dephosphorylation was calculated by subtracting values of stimulated cells of 100% and is shown as the mean ± sem. Asterisk indicates a significant (*, P < 0.05) difference to zero. Hashed sign indicates a significant (#, P < 0.05) difference between siRNA-1 and the control.