Figure 2

Molecular interaction between AMPK-α1 and TAK1. (a) The HEK293 cells were transfected with Myc-TAK1, Flag-AMPK-α1, or Myc-TAK1 and Flag-AMPK-α1 vector. At 36 h after transfection, cells were treated with or without LPS (100 ng/ml) for 45 min. The cells were extracted and immunoprecipitated with anti-Myc antibody. The interaction was detected by western blotting with anti-Flag antibody. The presence of Myc-TAK1 and Flag-AMPK-α1 in the pre-IP lysates was verified by western blot analysis. (b) The HEK293 cells were transfected with Flag-AMPK-α1 and wt Myc-TAK1, Flag-AMPK-α1 and Myc-TAK1 (1–500), Flag-AMPK-α1 and Myc-TAK1 (1–400), Flag-AMPK-α1 and Myc-TAK1 (1–300), Flag-AMPK-α1 and Myc-TAK1 (1–200), or Flag-AMPK-α1 and Myc-TAK1 (1–100) vectors. At 36 h after transfection, transfected cells were extracted and immunoprecipitated with anti-Flag antibody. The interaction was detected by western blotting with anti-Myc antibody. The presence of Myc-TAK1 mutants and Flag-AMPK-α1 in the pre-IP lysates was verified by western blot analysis. (c) The HEK293 cells were transfected with Myc-TAK1 and wt Flag-AMPK-α1, Myc-TAK1 and Flag-AMPK-α1 (1–312), or Myc-TAK1 and Flag-AMPK-α1 (1–392). At 36 h after transfection, transfected cells were extracted and immunoprecipitated with anti-Myc antibody. The interaction was detected by western blotting with anti-Flag antibody. The presence of Flag-AMPK-α1 mutants and Myc-TAK1 in the pre-IP lysates was verified by western blot analysis. (d) The THP-1 cells were treated with or without LPS (100 ng/ml) for 45 min. Cells were extracted and immunoprecipitated with anti-TAK1 antibody. The interaction was detected by western blotting with anti-AMPK-α1 antibody. The presence of TAK1 in the pre-IP lysates was verified by western blot analysis. (e) A model of molecular interaction between AMPK-α1 and TAK1. Above results suggest the molecular interaction between the autoinhibitory domain of AMPK-α1 and the N-terminus of TAK1. (f) WT THP-1, AMPK-α1^KD, and TAK-1^KD THP-1 cells were treated with or without LPS (10 ng/ml) for different times as indicated. Cell lysates were subjected to western blot analysis using the indicated antibodies. (g and h) WT THP-1, AMPK-α1^KD, and TAK-1^KD THP-1 cells were treated with or without LPS (10 ng/ml) for different times as indicated. In vitro kinase assay for TAK1 (g) and AMPK-α1 (h) was performed as described in Materials and Methods. Error bars indicate ±S.D. of triplicate samples. *P<0.05, **P<0.01