Insulin receptor substrate 1 irs 1

HL-1 cells transduced with adenoviral particles, or stimulated with HP/HI, were challenged with 200 nmol/l insulin for 30 min. To obtain whole cellular extracts RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Igepal, 0.5% Sodium deoxycholate, 0.1% sodium dodecyl dulfate (SDS) containing proteases and phosphatases inhibitors) was used. Protein concentration was measured by the BCA protein assay™ and equal amounts were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon polyvinylidene diflouride (PVDF) membranes. Western blot analyses were performed using antibodies against phospho-Ser 473 -AKT, AKT (Cell Signaling), phospho-Thr 642 -AS160 (AKT substrate of 160 kDa), AS160 (Upstate), Insulin receptor substrate 1 (IRS-1) (Cell Signaling), succinate dehydrogenase complex iron sulfur subunit B Complex II (CII-SDHB) (Abcam) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling). Detection was performed using the appropriate horseradish peroxidase-labelled IgG and the Chemiluminescent Peroxidase Substrate-1 (Sigma). The size of detected proteins was estimated using protein molecular-mass standards (Thermo Scientific, Waltham, MA USA). Western blot images were analysed with a Molecular Imager (ChemiDoc XRS, BioRad) and quantified with Quantity One® (BioRad).