Quantifying Insulin-Stimulated AKT Activation

Primary hepatocytes were stimulated with the indicated stimulant for 3 h and then 100 nM insulin and stimulant were added 10 min prior procession in serum free William’s Medium E. Lysates of primary hepatocytes were prepared in a modified RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.6, 1% Triton X-100, 0.5% Sodium deoxycholate and 0.1% SDS) supplemented with cOmplete^TM protease inhibitor (Roche, Mannheim, Germany) and a protein phosphatase inhibitor set (Sigma-Aldrich, Steinheim, Germany) described by Titchenell et al. [82 (link)]. Successful lysis was ensured by microscopic inspection. Supernatants were extracted from cellular debris following centrifugation (15,000× g, 10 min). The AKT activation was then assessed with Phospho-AKT(pSer473)/pan-AKT ELISA kit (Sigma-Aldrich, Steinheim, Germany) according to the manufacturer’s instruction and protein content was assessed using a BCA protein assay kit (Sigma-Aldrich, Steinheim, Germany).