Fig. 4

Activation of AMPK ablates the interaction between Insig and the ubiquitin ligase gp78. a, b Metformin reverses the gp78-mediated degradation of Insig-1 (a) and Insig-2 (b). myc-Insig-1 or FLAG-Insig-2 was co-transfected with myc-gp78 in HEK293 cells for 24 h, followed by treatment with 2 mM metformin for 24 h. Immunoblots were performed. c, d AMPK-specific agonist A769662 inhibits the interaction between Insig and gp78. FLAG-tagged Insig-1 (c) or Insig-2 (d) was co-transfected with myc-tagged gp78 in HEK293 cells for 24 h, followed by treatment with 100 μM A769662 for 8 h. The cell lysates were incubated with FLAG antibodies and purified with protein A/G-Sepharose beads. The precipitates and lysates were individually immunoblotted with indicated antibodies