Phosphorylated insulin receptor substrate 1

Whole retinal lysates were collected into lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were placed into pre-cast trisglycine gels (Invitrogen, Carlsbad, CA), and blotted onto nitrocellulose membranes. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) with 5% BSA, membranes were treated with a phosphorylated insulin receptor (Tyr 1150/1151), insulin receptor, phosphorylated Akt (Ser473), total Akt, phosphorylated insulin receptor substrate 1 (Ser307), total IRS-1 (Cell Signaling Technology, Danvers, MA) TNF, (Abcam, Cambridge, MA), and beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies overnight. The following day, membranes were incubated with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were visualized using Chemiluminescence (Thermo Scientific, Pittsburgh, PA). Data was analyzed on an Azure C500 machine (Azure Biosystems, Dublin, CA). Western blot band densities were measured using Image Studio Lite software.

Whole retinal lysates from mice were collected into lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated onto a pre-cast tris-glycine gel (Invitrogen, Carlsbad, CA), and blotted onto nitrocellulose membrane. After blocking in TBST (10mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, the membranes were treated with TLR4 (Abcam, Cambridge, MA), Akt, phosphorylated Akt (Ser473), insulin receptor, phosphorylated insulin receptor (Tyr 1150/1151), insulin receptor substrate 1, phosphorylated insulin receptor substrate 1 ((Ser 307), Cell Signaling Technology, Danvers, MA)) and beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected by chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA) and data was acquired using an Azure C500 (Azure Biosystems, Dublin, CA). Western blot data were assessed using Image Studio Lite software.