Supersignal west femto luminol enhancer solution

Muscle tissue samples were homogenized in a Tris lysis buffer with the use of Qiagen Tissuelyser. Proteins were separated on a 4–15% Criterion TGX Precast Gel (Bio-Rad) by electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad), using the Trans-Blot Turbo Transfer system at 2.5A–25V for 10 min (Bio-Rad). After blocking with 1% Fish Skin Gelatin (Sigma-Aldrich), the membrane was incubated overnight at 4°C with either anti-Akt (Cell Signalling), anti-phospho-Akt (Ser473, Cell Signalling), anti-mTOR (Cell Signalling) or anti-phospho-mTOR (Ser2448, Cell Signalling) antibodies in 1% Fish Skin Gelatin. Secondary anti-bodies used were HRP-conjugated Polyclonal Goat Anti-Rabbit Immunoglobulins (DAKO) in 1% Fish Skin Gelatin, and proteins were visualised with super signal west femto luminol/enhancer solution (Thermo Scientific). Proteins were analysed by the ChemiDoc XRS system (Bio-Rad) and the Quantity One software (Bio-Rad). Unspecific staining of proteins with Reactive Brown (Sigma-Aldrich) was used as loading and transfer control.

Cultures were grown at 37 °C with shaking for 16 h, then back-diluted (1:50) into 5 mL of LB containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol and incubated as before until an OD₆₀₀ of ~ 0.3 to 0.5 was reached. Expression of the coding sequence was induced with 0.05 mM IPTG for 2 h at 30 °C. A volume of cells corresponding to an OD₆₀₀ of either 0.02 or 0.2 was harvested by centrifugation then resuspended in 2 × Laemlli loading buffer [125 mM Tris–HCl pH 6.8, 4% SDS, 3% Glycerol, 0.02% bromphenol blue, 20% β–mercaptoethanol]. Proteins were separated by 12% SDS-PAGE then transferred to a nitrocellulose membrane using a semi-dry transfer apparatus (Bio-Rad, USA). The nitrocellulose membranes were probed with an antibody against either β-lactamase (Thermo Scientific, USA) or the poly-histidine tag (His-Probe, ThermoFisher Scientific, USA). Binding was detected using anti-mouse IgG linked to horseradish peroxidase (GE healthcare, USA) and a SuperSignal West femto luminol/enhancer solution (ThermoFisher Scientific, USA). Luminescence emitting from the nitrocellulose membrane was detected using an Azure Biosystems c600 device.

For western blotting analyses, whole cell lysates containing equal amounts of protein (25 μg) from corneal tissue were separated on 7.5% acrylamide gel by SDS-PAGE. After electrophoresis, separated proteins were transferred to polyvinylidene difluoride membranes (Invitrogen, Carlsbad, CA, USA) and incubated with anti-NF-kB (Thermo Scientific) and anti-β-actin (Sigma, St Louis, MO, USA) in order to confirm equal loading of each protein. Bound antibody was detected using appropriate HRP-conjugated second antibodies (Promega, Madison, WI, USA) for more than 60 min. Membranes were then washed and developed with SuperSignal West Femto Luminol/Enhancer solution (Thermo Scientific). Immunoreactivity on blots was detected using a LAS-4000 Luminescent Image Analyzer with CCD Camera (Fujifilm, Tokyo, Japan) and quantified, with all results expressed as ratios to β-actin protein amounts.