Insulin receptor phosphorylated on tyr960

After rinsing with cold PBS, REC were collected in a lysis buffer containing protease and phosphatase inhibitors and scraped into tubes. Equal amounts of protein were separated on precast tris-glycine gels (Invitrogen, Carlsbad, CA), and then blotted onto a nitrocellulose membrane. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, the membrane was treated with the appropriate primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected by chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA). Primary antibodies used were phosphorylated Akt (Serine 473), Akt, SOCS3, insulin receptor (all purchased from Cell Signaling, Danvers, MA), phosphorylated insulin receptor (tyrosine 1150/1151) (Enzo Life Sciences), insulin receptor phosphorylated on Tyr960 (Cell Applications, San Diego, CA), and beta actin (Santa Cruz, Santa Cruz, CA).

Equal amounts of protein from the tissue extracts were separated on the pre-cast tris-glycine gel (Invitrogen, Carlsbad, CA, USA), blotted onto a nitrocellulose membrane. After blocking in TBST (10 mM Tris–HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, the membrane was treated with appropriate primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected by a chemiluminescence reagent kit (Thermo Scientific, Waltham, MA, USA). Primary antibodies used were phosphorylated Akt (Serine 473), Akt, Bax, Bcl-xL, Cytochrome C, SOCS3, phosphorylated insulin receptor (tyrosine 1150/1151), insulin receptor (all purchased from Cell Signaling, Danvers, MA, USA), insulin receptor phosphorylated on Tyr960 (Cell Applications, San Diego, CA, USA), and beta actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).