Chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Hanover Park, IL) unless otherwise noted. The reagents and apparatus for SDS-PAGE and nonfat dry milk (no. 170-6404) were obtained from Bio-Rad (Hercules, CA). Pierce MemCode Reversible Protein Stain Kit (#24585), Bicinchoninic acid protein assay (#23225), Tissue Protein Extraction Reagent (TPER; #78510), Protein G magnetic beads (#10004D), and DynaMag
TM-2 magnet (#12321D) were from Thermo Fisher Scientific (Waltham, MA). Anti-phospho Akt Ser
473 (pAkt
Ser473; #9271), anti-phospho Akt Thr
308 (pAkt
Thr308; #13038), anti-Akt (#4691), anti-phospho AS160 Thr
642 (pAS160
Thr642; #8881), anti-phospho AS160 Ser
588 (pAS160
Ser588; #8730), anti-phospho AMPKα Thr
172 (pAMPKα
Thr172; #2531), anti-AMP-activated protein kinase-α (AMPKα; #5831), anti-acetyl CoA carboxylase (ACC; #3676), anti-phospho ACC Ser79 (pACC
Ser79; #3661), anti-TBC1D1 (#5929), anti-hexokinase II (HKII; #2867), anti-insulin receptor (IR; #3025), anti-α-Tubulin (#2144), anti-Na
+/K
+-ATPase (#3010) and anti-rabbit IgG horseradish peroxidase conjugate (#7074) were from Cell Signaling Technology (Danvers, MA). Anti-phospho TBC1D1 Thr590 (pTBC1D1
Thr590; #AF2422) was from Sapphire North America (Ann Arbor, MI). Anti-phospho AS160 Ser
704 (pAS160
Ser704) was provided by Dr. Jonas Thue Treebak (Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Denmark). Anti AMP-activated protein kinase γ3 (γ3-AMPK) was provided by Dr. David Thomson (Brigham Young University, USA) (
Hardman et al. 2014). Anti-Akt Substrate of 160 kDa (AS160; #ABS54), Anti-GLUT4 (GLUT4; #CBL243), Anti-phospho-TBC1D1 Ser237 (pTBC1D1
Ser237; #07-2268), P81 Phosphocellulose Squares (#20-134) and enhanced chemiluminescence Luminata Forte Western HRP Substrate (#WBLUF0100) were from MilliporeSigma (Billerica, MA). Anti-HMGCR (HMGCR, #BS-5068 R) and anti-phospho HMGCR Ser872 (pHMGCR
Ser872, # BS-4063 R) were from Bioss Antibodies (Woburn, MA). Anti-ABCA1 (ABCA1, #NB400-105SS) was from Novus Biologicals (Littleton, CO). Anti-phospho ABCA1 Ser2054 (pABCA1
Ser2054; #ab12064) was from Abcam (Cambridge, MA). 3-
O-methyl-[
3 H] glucose ([
3 H]3-MG) was from Sigma-Aldrich, and [
14C] mannitol was from PerkinElmer (Boston, MA). [γ-
33 P]-ATP was from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Liquid scintillation cocktail (#111195-CS) was from Research Products International (Mount Prospect, IL).