Precast tris glycine gels

As described in our previous study (16 (link)), REC were rinsed with cold PBS, collected in 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 (10mM 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 chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA). Primary antibodies used were phosphorylated STAT3 (Tyr705) and total STAT3 (purchased from Cell Signaling, Danvers, MA), VEGF (Abcam, Cambridge, MA), ADAM10, ADAM17 (Santa Cruz Biotechnology, Dallas, TX), and beta actin (Santa Cruz, Santa Cruz, CA).

To analyze total TRAF3IP2 and THEMIS protein levels, T cell blasts were washed in PBS and lysed in 1× Triton lysis buffer with complete protease inhibitor cocktail (Roche, Basel, Switzerland) on ice for 20 minutes. The lysates were then cleared at 14,000 × g at 4°C for 15 minutes. Lysates were then diluted with 2X SDS sample buffer (Quality Biologicals, Gaithersburg, MD) supplemented with 10% BME. Approximately 2 × 10⁶ cell equivalents were separated by SDS-PAGE on 4% to 20% precast tris-glycine gels (Invitrogen) and transferred to a nitrocellulose membrane (Invitrogen). Membranes were blocked with 3% BSA in Tris-buffered saline with 0.01% Tween-20 (TBST) for 30 minutes at room temperature before incubating with primary antibody overnight at 4°C. After 3× 5-minute washes with TBST at room temperature with rocking, fluorescent secondary antibody was added for 2 hours at room temperature. After 5× 5-minute washes in TBST and 1 wash in PBS, membranes were analyzed on the LI-COR Odyssey imaging system or the Azure c300 imaging system.

Whole retinal lysates or REC samples were rinsed with cold PBS, collected in 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 (10mM 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. Primary antibodies used were phosphorylated insulin receptor (Tyr 1150/1151), total insulin receptor, phosphorylated Akt (Ser473), Akt, phosphorylated IRS-1 (Ser307), total IRS-1 (Cell Signaling Corp, Danvers, MA), and beta actin (Santa Cruz, Santa Cruz, CA). Antigen-antibody complexes were detected using a chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA). Mean densitometry of immunoreactive bands was assessed using a C500 (Azure Biosystems), and results were expressed in densitometric units and compared to control groups for each individual experiment. For the phosphorylated antibodies, the ratio of phosphorylated to total protein levels are presented.

30–60μg of protein were separated on precast tris-glycine gels (Invitrogen) and transferred to PVDF membrane. Membranes were blocked with 5% BSA/TBST. Primary antibodies, after incubation in 5% BSA/TBST, were detected using HRP conjugated secondary antibodies in chemiluminescent solution using the Quantity One imaging software on a Bio-Rad Gel Docking system. Primary antibodies: Rabbit mAb Bnip3 (EPR4034) from Abcam, rabbit anti-P-AktSer473, rabbit mAb P-Akt308 (C31E5E), rabbit mAb Akt (pan) (C67E7), mouse mAb Histone H3 (96C10), rabbit mAb P-Ser555 ULK (D1H4, #5869), rabbit mAb ULK (D8H5, #8054), rabbit mAb P-AMPKα (Thr172) (40H9, #2535), and rabbit anti-AMPKα (#2532) from Cell Signaling Technology, rabbit anti-LC3B (NB100–2200) from Novus Biologicals, mouse mAb GAPDH from Millipore, rat anti-integrin α6 (GoH3) and mouse anti-HIF1α from BD Pharmingen, mouse mAb AR (441) from Santa Cruz, mouse anti-α tubulin and β-actin-HRP mouse mAb from Sigma-Aldrich), and rabbit anti-integrin α6 (AA6A, A6NT).^{12 (link), 35 (link)}

Uninfected and infected corneas were excised at 3 and/or 5 days p.i. from all treatment groups of B6 and BALB/c mice. Individual corneas were homogenized in 250 μL RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA), sonicated to promote further lysis and centrifuged at 12,000 RPM for 20 minutes. For Western blot analysis, supernatants were collected and normalized for equal amounts of protein, then separated on pre-cast tris-glycine gels (Invitrogen, Carlsbad, CA, USA) and 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) fat free milk powder, membranes were treated with the following primary antibodies: CysLT₁ (Abcam, San Francisco, CA, USA), CysLT₂ (Invitrogen, Carlsbad, CA, USA), total/phosphorylated-Erk1/2, NF-κB p65, NF-κB p105/p50 (Cell Signaling Technologies, Danvers, MA, USA), and β-actin (Santa Cruz, CA, USA); then followed by incubation with secondary antibodies (Santa Cruz) labeled with horseradish peroxidase. All primary antibodies were used at a dilution at 1:500 and secondary antibodies at a 1:1000 dilution. Images were collected (Azure Biosystem C500, Dublin, CA) and target protein levels were quantified (Image Studio Lite software) after normalizing to β-actin.