Figure 5. PPARα co-activation by AMPKα is independent of the LXXLL domain of AMPKα.

(A) 293 cells were transfected with the reporter plasmid rAOX(PPRE)-CAT and the expression plamid for RXRα (0.03 μg), and co-transfected with the expression plasmid for mPPARα (0.03 μg), hAMPKα2 (5.0 μg) or hAMPKα2-(313–552) (5.0 μg) as indicated. Following transfection, cells were incubated for 17 h in the presence of 20 μM nafenopin. Fold induction represents CAT activity (means±S.E.M. of duplicate plates differing by no more than 10% from five independent experiments) normalized by CMV-β-galactosidase and further normalized by CAT activity of pSG5-transfected cells. Significance: *P<0.05 compared with no added hAMPKα2. Expression of hAMPKα2 and hAMPKα2-(313–552) was verified by Western blot analysis of the cell lysate using sheep anti-hAMPKα2 antibody (inset): lane 1, pcDNA3 (5.0 μg); lane 2, hAMPKα2 (5.0 μg); lane 3, hAMPKα2-(313–552) (5.0 μg). Kd, kDa. (B) COS-7 cells were transfected with the reporter plasmid rAOX(PPRE)-CAT and the expression plasmid for mPPARα (0.05 μg), and co-transfected with hAMPKα2 (2.0 μg) or hAMPKα2(L²⁰⁴YAAA) (2.0 μg) as indicated. Following transfection, cells were incubated in the presence of 10 μM nafenopin. Fold induction represents CAT activity (means±S.E.M. for triplicate plates or means of duplicate plates differing by no more than 10%) normalized by CMV-β-galactosidase and further normalized by CAT activity of pSG5-transfected cells. Significance: *P<0.05 compared with no added AMPKα.