Western Blot Analysis of AMPK

Western blot analysis was performed as previously described [22 (link)]. Whole animal protein extract prepared from 300 gravid adult hermaphrodites from each treatment was used per gel well. Antibodies bound to a nitrocellulose membrane (PROTRAN BA83, Whatman, Sigma-Aldrich, St. Louis, MO, USA) were visualized using an ECL Western blotting detection kit (Amersham, GE Healthcare Life Sciences, Pittsburgh, PA, USA), and the respective band intensities were measured using a LAS-3000 image analyzer with Multi Gauge (v.3.0) (Fuji Film, Tokyo, Japan). The following primary and secondary antibodies were used: rabbit anti-AMPK (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pAMPK (1:1000, Cell Signaling Technology, Danvers, MA, USA), mouse anti-α-tubulin (1:1000; Sigma-Aldrich, St. Louis, MO, USA), HRP-conjugated goat anti-rabbit IgG (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), and HRP-conjugated donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch, West Grove, PA, USA).