Fig. 3.

Administration of AMPK agonist represses the ubiquitination and degradation of Insig. a, b AMPK agonist decreases the ubiquitination levels of Insig-1 and Insig-2. Plasmids encoding myc-tagged Insig-1 (a) or FLAG-tagged Insig-2 (b) were co-transfected with HA-tagged ubiquitin (HA-Ub) for 24 h, followed by treatment with 100 μM A769662 for 4 h. The cell lysates were incubated with myc or FLAG antibodies and purified with protein A/G-Sepharose beads. The precipitates and lysates were individually immunoblotted with indicated antibodies. c, d AMPK agonists reduce the degradation rate of Insig-1. HEK293 cells stably expressing FLAG-tagged Insig-1 were treated with 100 μM A769662, 2 mM metformin, and 1 mM AICAR, followed by incubation in the medium containing 100 ng/ml cycloheximide for the indicated time. Data are presented as the mean ± SEM, n = 4