Total ampk

JC105 was synthesized in our laboratory (see below). Isosteviol was purchased from Sigma–Aldrich Co. Ltd. U0126 was purchased from Cell Signaling Technology. 3‐[4, 5‐dimethylthiazol‐2‐yl] 2, 5‐diphenyl‐tetrazolium bromide (MTT) was purchased from VWR Life Sciences. Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), 0.05% Trypsin‐EDTA (1×), penicillin (10 000 U/mL)‐streptomycin (10 000 µg/mL) (Pen Strep), phosphate‐buffered saline (PBS, 1×), no glucose and no sodium pyruvate DMEM were purchased from Gibco. RIPA buffer was obtained from Biosharp. Protease inhibitor (100x) was obtained from Beyotime. Bicinochoninic acid (BCA) assay kit was obtained from Beyotime. Stock concentration of JC105 (100 mM) and U0126 (10 mM) was dissolved in DMSO. MTT (5mg/ml) was dissolved in DMEM. During all treatments, the concentration of DMSO was below 0.1% (v/v). Phospho‐ERK 1/2 (Thr²⁰²/Tyr²⁰⁴), Total‐ERK1/2, Phospho‐DRP1 (Ser⁶¹⁶), Total‐DRP1, Phospho‐AKT (Ser⁴⁷³) and Total‐AKT were obtained from Cell Signaling Technology. Phospho‐AMPKα (Thr¹⁷²) and Total‐AMPK were obtained from Abcam and GAPDH was obtained from Proteintech. Anti‐rabbit IgG and anti‐mouse IgG were obtained from Proteintech. Enhanced chemiluminescence reagent was obtained from Affinity Biosciences.

AICAR and 4‐Nitrobenzylthioinosine (NBMPR) were purchased from Tocris (Abingdon, UK). Dibutyryl cAMP, uridine and metformin were purchased from Sigma (Dorset, UK). LC‐MS materials were obtained from Fisher Scientific (Loughborough, UK) unless otherwise stated. Glucagon was obtained from Novo Nordisk (Bagsvaerd, Denmark). 8CPT‐cAMP was purchased from Calbiochem (Nottingham, UK) and 8CPT‐2‐Methyl‐O‐cAMP (8CPT‐2MeO‐cAMP) was purchased from Biolog Life Sciences Institute (Bremen, Germany). Donkey anti‐goat 680RD, donkey anti‐mouse 680RD, donkey anti‐rabbit 800CW secondary antibodies were purchased from LI‐COR Biosciences, UK. The GAGO glucose oxidase kit was purchased from Sigma. [5,6‐³H] uridine was purchased from Perkin Elmer (Beaconsfield, UK). pAMPK (Thr172) and total ACC antibodies were purchased from Cell Signalling Technologies (Leiden, UK). Total AMPK was purchased from Abcam (Cambridge, UK) and actin was purchased from Proteintech (Manchester, UK). pACC was made in‐house by Division of Signal Transduction Therapy (University of Dundee).