Ecl select western blotting detection reagent

Cells and EVs were lysed in RIPA buffer (50 mM Tris, pH 8.0, 0.6 M NaCl, 4% Triton X-100, 2% sodium deoxycholate, 0.1% SDS). Ten micrograms of cellular and EV total protein were applied per lane and separated by 10% SDS-PAGE. Proteins were electroblotted onto nitrocellulose membranes and stained with Ponceau S solution to check the protein loading. The membranes were destained, blocked with 10% (w/v) fat-free milk and then incubated with the primary antibodies: CD9 (Santa Cruz, Germany) (1:500), ALIX (Santa Cruz, Germany) (1:1000), TSG101 (Abcam, UK) (1:1000), β-actin (Abcam, UK) (1:4000) and Calnexin (Abcam, UK) (1:1000). After washing, the membranes were incubated with peroxidase - conjugated rabbit anti-mouse secondary antibody (Santa Cruz, Germany) (1:2000) or mouse anti-rabbit secondary antibody (Santa Cruz, Germany) (1:2000), washed and processed with ECL Select Western Blotting Detection Reagents (GE Healthcare, USA) according to manufacturer’s instructions.

Extracted proteins were loaded onto a SDS–polyacrylamide gel electrophoresis (15%) and transferred onto a nitrocellulose membrane (AmershamTM Protran TM; GE Healthcare, Germany). Proteins were revealed by staining the membrane with Ponceau S dye (Sigma‐Aldrich). After incubation in blocking buffer (10 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20, 5% milk powder), the membranes were incubated overnight at 4°C with primary antibody. Primary antibody (see the reference below) was then removed and a 1 h incubation was done with horseradish peroxidase‐conjugated mouse or rabbit secondary antibody (1:10 000 or 1:5000 dilution, respectively; Jackson ImmunoResearch Laboratories, Baltimore, MD). Immunoblots were visualized on an ImageQuant LAS4000 system (GE Healthcare, Buckinghamshire, UK) using the ECL Select™ Western Blotting Detection Reagents (GE Healthcare). Western blot images are illustrated in FiguresS1 and S2.

Total protein was extracted from cultured ARPE-19 cells using NP40 cell lysis buffer (Cat#FNN0021, Invitrogen, Frederick, MD), and the concentration was calculated by DC protein assay (Cat#500–0016, Bio-Rad, Philadelphia, PA). Equivalent amounts of extracted protein were loaded into each well and electrophoresed on a Nupage 4–12% Bis-Tris gel (Cat#NP0335BOX, Novex, Carlsbad, CA), then transferred to a 0.2μm PVDF membrane (Cat#ISEQ00010, Millipore, Billerica, MA), blocked with non-fat dry milk (Cat#9999, Cell signaling, Danvers, MA) and incubated with primary antibodies against MnSOD (Cat#16956, Abcam, Cambridge, MA) at 1:2000, TrxR₂ (Cat#sc-46278, Santa Cruz Biotechnology, Dallas, TX) at 1:250, and GAPDH (Cat#5174, Cell Signaling Technology, Danvers, MA) at 1:1000, for 2 hours. The membrane was washed 3 times for 10 minutes each time and incubated with secondary antibodies at 1:10000 for 1.5 hours. After washing, membranes were incubated with the chemiluminescent substrate (Cat#RPN2235, ECL Select western blotting detection reagents, GE Healthcare Life Sciences, Piscataway, NJ) for 5 minutes. Band signals were detected by an image-scanning densitometer (ChemiDoc imaging system; Bio-Rad) and quantitated by Image J 2.0.

Western blotting was performed according to the method described previously [17 (link)]. In brief, protein extracts (10 µg) were separated by SDS/polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were then hybridized with antibodies against AKT (Cell Signaling Technology 9272; 1/500) and phospho-AKT (Ser473, Cell Signaling Technology 9271, 1/500; Thr308, Cell Signaling Technology 9275, 1/500). Immunoreactivity was detected with ECL Select Western Blotting Detection Reagents (GE Healthcare Life Science, Marlborough, MA, USA) and imaged by Image Quant LAS 500 (GE Healthcare Life Science). An antibody against β-actin (C4) (Santa Cruz Biotechnology sc-47778; 1/5000) was used as an internal control. The band signal obtained was quantified using Image J and the ratio of p-AKT to β-actin expression was calculated. The whole images of Western blotting are shown in Supplementary Figure S1.

RPE cells (2.5 × 10⁵/well, 6-well plate) were seeded and cultured for 3 days. Before treatment with TNF-α and/or AICAR, cells were serum-starved for 24 hours in serum-free medium. After treatment, cells were washed with cold PBS and lysed in NP40 cell lysis buffer (Invitrogen, Carlsbad, CA) containing protease and phosphatase inhibitor cocktail (Roche, Indianapolis, IN). Samples were loaded onto a NuPAGE 4–12% Bis-Tris Gel (Novex, Carlsbad, CA), transferred to a polyvinylidene difluoride (PVDF) membrane (0.45 μm; Millipore, Billerica, MA), blocked with 5% non-fat dry milk, and incubated with appropriate primary antibodies. Blots were subsequently incubated with secondary antibodies and images were developed using chemiluminescent substrate (ECL Select western blotting detection reagents, GE Healthcare Life Sciences, Piscataway, NJ). Band signals were detected by an image-scanning densitometer (ChemiDoc imaging system; Bio-Rad) and quantitated by ImageJ 2.0.

The heart tissue was homogenized in RIPA buffer containing NaF, trypsin inhibitor, leupeptin, glycerophosphate, and orthovanadate. Samples of heart tissue lysate were resolved on SDS-PAGE according to a standard protocol. After being transferred to the membranes, the samples were immunoblotted with primary antibodies, followed by secondary antibodies conjugated to a horseradish peroxidase. Bands were revealed using ECL select western blotting detection reagents (GE Healthcare, Buckinghamshire, UK), and band density was quantified using the Image J software. The following primary antibodies were used: CD36 (#sc-9154) and actin antibodies (#sc-1616) were obtained from Santa Cruz biotechnology (Santa Cruz, CA). GLUT-4 antibody (#2213), cleaved caspase-3 antibody (#9661), phospho-AMPKα (Thr172) antibody (#2535), and LC3A/B (1/2) antibody (#12741), were purchased from Cell Signaling (Danvers, MA).