Phospho as160

Cells were homogenized in a buffer containing 50 mM Tris HCl, 150 mM NaCl, 5 mM Ethylenediaminetetraacetic acid (EDTA), 0.1% Sodium dodecyl sulfate (SDS), 1% sodiumdeoxycholate and protease and phosphatase inhibitor cocktails (Sigma, Schnelldorf, Germany). After sonication and centrifugation, protein concentration was measured with the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific). A total of 20 µg of protein were loaded on an SDS-PAGE gel and transferred to a nitrocellulose membrane. Following blocking, filters were incubated with antibodies directed against His, MBP, phosphor–AMPK (Thr172), alpha–AMPK, phospho–AS160 (Thr642), phospho–Akt (Thr308), Akt, ACC, phospho–ACC (all Cell Signaling Technology, Europe B.V., Frankfurt am Main, Germany), GLUT1, GLUT4 (both from Cushman, S.W. [32 (link)]), MBP (NEB), AS160 (Merck Millipore, Darmstadt, Germany) and GAPDH (Abcam, Cambridge, UK). After incubation with peroxidase-conjugated secondary antibody, blots were subjected to the enhanced chemiluminescent detection method with the Fusion FX7 imaging system (Peqlab, Erlangen, Germany).

Trypsin solutions, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), antibiotic/antimycotic, and horse serum were purchased from GIBCO Life Technologies (Gaithersburg, MD, USA). We obtained 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and Dimethyl sulfoxide (DMSO) from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). We used a Mem-PER Plus Membrane Protein Extraction Kit and 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] Amino)-2-deoxyglucose (2-NBDG) from Thermo (Sunnyvale, CA, USA). Protease inhibitor cocktail, phosphatase inhibitor cocktail, Compound C, and STO-609 were obtained from Sellbeck chemicals (Houston, TX, USA). Our siRNA was purchased from Shanghai GenePharma company (Shanghai, China). We utilized antibodies against phospho-AKT (Ser473), AMPKα (Thr172), phospho-AKT (Thr308), phospho-AS160 (Ser588), phospho-p38 MAPK (Thr180/Tyr182), phospho-ACC (Ser79), phosphoinositide 3-kinase (PI3K, P110β), AS160, AMPKα, and ACC from Cell Signaling Technology (Danvers, MA, USA); AKT, ATP1A1, p38 MAPK, and β-actin from Proteintech (Wuhan, China); and GLUT4 from Abcam (Cambridge, UK). Secondary antibodies and insulin were sourced from Yeasen Biotech (Shanghai, China). A glucose assay kit was procured from Shanghai Kexin Biotechnology Research Institute (Shanghai, China).

Immunoblotting was performed with the following commercially available antibodies: AS160, phospho-AS160, Akt, phospho-Akt, IRS-1, phospho-IRS-1 (Ser307, Ser612, Ser1101), AMPK, phospho-AMPK, Na/K ATPase, GAPDH, anti-goat IgG, anti-rabbit IgG, and anti-mouse IgG from Cell Signaling Technology (Beverly, MA, USA). Phospho-IRS-1 (Tyr 989), and PI3-kinase p85α, Tyrosine were from Santa Cruz Biotechnology. β-actin was obtained from Sigma (St. Louis, Mo, USA).