Figure 6

Pharmacological activation of AMPK leads to dephosphorylation and nuclear localization of TFEB. A) AMPKα1α2^+/+ or AMPKα1α2^−/− MEFs were treated with vehicle (DMSO), 10 µM 991, or 2 mM AICAR for 1 h. Cytoplasmic and nuclear fractions were prepared and lysates (20 µg) from the fractions were used for Western blot analysis of the indicated proteins. B) COS1 cells were transfected for 48 h with no vector (negative control) or the vector containing Flag-tagged TFEB WT or S142A mutant construct. The WT or mutant TFEB was immunoprecipitated (IP) from cell lysate (500 μg) with 1 μg of anti-Flag antibody and the immunoprecipitates were subjected to Western blot analysis, with total or phospho-S142 (pS142) TFEB antibodies. Images are representative of n = 2. C) MEFs were treated with AMPK activators (10 µM 991 or 2 mM AICAR) or various concentrations of mTOR inhibitor rapamycin (0.05, 0.1 and 0.5 µM) for 1 or 4 h. Cell lysates (20 µg) were subjected to Western blot analysis using the indicated antibodies. D) The experiment was performed as described in C, except Torin-2 (5 or 10 nM) was used as an alternative mTOR inhibitor. Images are representative of n = 2.