Tbc1d1

To measure AMPK signaling, EDL muscle fibers were incubated in an oxygenated Buffer A bath for 5 min. Buffer A was aspirated and replaced with fresh Buffer A containing DMSO (vehicle), PF-739 (10 μM). A subset of muscle fibers treated with vehicle were contracted by electrical pulsation for 30 min. EDL muscle fibers were stored in a tube and immediately snap frozen in liquid nitrogen. For western blotting, EDL muscle fibers were homogenized in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% IGEPAL, 0.5% w/v sodium deoxycholate and 0.1% w/v sodium dodecyl sulfate) with 1 mM phenylmethylsulfonyl fluoride (Thermo Fisher Scientific), protease inhibitor cocktail (Sigma) and Halt’s phosphatase inhibitor (Thermo Fisher Scientific). Protein samples were subjected to SDS-PAGE using a 4–15% gradient gel (Bio-Rad), and transferred to a nitrocellulose membrane. Membranes were incubated overnight with primary antibodies against phospho-AMPK (Thr172) (Cell Signaling, Cat# 2535S), phospho-ACC (Ser79) (Millipore, Cat#07–303), phospho-TBC1D1 (Millipore Cat#07–2268), pan-AMPKα (Cell Signaling, Cat #5831S), ACC (Cell Signaling, Cat#3662S), TBC1D1 (Cell Signaling, Cat#4629S), and α-tubulin (Cell Signaling, Cat#2125S), followed by HRP-conjugated secondary antibodies and chemiluminescent detection.