Western lightning plus enhanced chemiluminescence

Reagents, molecular weight markers, and nitrocellulose membrane were purchased from BioRad (Mississauga, ON, Canada). Western Lightning Plus enhanced chemiluminescence (ECL) was purchased from Perkin‐Elmer (NEL105001EA). The following primary antibodies were purchased from Cell Signaling: phospho‐AKT Ser⁴⁷³ (Cat. # 4060), phospho‐AKT Thr³⁰⁸ (Cat. # 9275S), phospho‐CaMKII Thr²⁸⁷ (Cat. # 12716), phospho‐STAT5b Tyr⁶⁹⁴ (Cat. # 4322), total AKT (Cat. # 9272S), total AMPK (Cat. # 2603S), total CaMKII (Cat. # 4436S) and total STAT5 (Cat. # 9363T). NP40 cell lysis buffer was acquired from Life Technologies and PMSF and protease inhibitor cocktail were obtained from Sigma (Cat. # 78830 and 9599). Insulin (Humulin rDNA origin) was purchased from Eli Lilly (Toronto, ON, Canada).
Incubation buffer (Krebs–Henseleit base) constituents were purchased from Sigma‐Aldrich and include: d‐glucose (Cat. #G‐8270), d‐mannitol (Cat. #M‐9546), sodium pyruvate (Cat. #P8574) and 3‐methyl‐O‐glucopyranose (Cat. #M‐4879). Growth hormone was obtained from Abcam (Cat. #ab68388). Acylated (Cat. #H‐4862) and unacylated (Cat. #H‐6264) ghrelin were sourced from Bachem (Torrance, CA). Radioactive ¹⁴C‐mannitol (Cat. #CFA‐238 – 250 μCi/1250 μL) and 3‐O‐[³H]methyl‐d‐glucose (Cat. #ART‐126 – 1 mCi/1000 μL) tracers were purchased from American Radiolabeled Chemicals (St. Louis, MO).

Retinal tissue was harvested 24 h after IRI to investigate protein expression. Total protein from 75% of the retina was extracted and processed for Western blotting, as previously described [56 (link),57 (link)]. The membranes were blocked with 5% skim milk or bovine serum albumin in Tween20/PBS to minimize non-specific antibody binding and were subsequently incubated overnight with the following protein-specific antibodies, diluted as recommended by the manufacturer (iNOS#130246, VEGF#102643, ICAM-1#100450, caspase 3 #110543, cleaved caspase #86952, all Genetex, Irvine, CA, USA; ß-actin #3700, p-p38 #4511, p38 8690, p-ERK1/2 #9101, ERK1/2 #4695, all Cell Signaling Technology, Danvers, MA, USA). After incubation with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Freiburg, Germany), the proteins were visualized with an enhanced chemiluminescence Western blotting detection reagent (Western Lightning plus enhanced chemiluminescence, #NEL104001EA, PerkinElmer, Waltham, MA, USA), according to the manufacturer’s instructions to visualize the proteins. Relative changes in protein expression in retinas exposed to IRI in the four treatment groups were calculated based on the untreated retinas of each respective animal. The chemiluminescence imaging system Fusion Fx^® (Vilber, Collegién, France) was employed for recordings and densitometric analysis.

Reagents, molecular weight markers, and nitrocellulose membrane were purchased from BioRad (Mississauga, ON, Canada). Western Lightning Plus enhanced chemiluminescence (ECL) was purchased from PerkinElmer (NEL105001EA). The following primary antibodies were purchased from Cell Signaling: phospho-Akt Thr³⁰⁸ (cat. # 9275), phospho-Akt Ser⁴⁷³ (cat. # 9271), and Akt pan (cat. #4691). Antibodies against cytochrome oxidase complex IV (COXIV; cat. #16056), citrate synthase (CS; cat. #96600), and antiubiquinol-cytochrome C reductase core protein 1 (core1; cat. 110252) were acquired from Abcam. NP40 cell lysis buffer was acquired from Life Technologies and PMSF and protease inhibitor cocktail were obtained from Sigma (cat. #78830 and 9599). Insulin (Humulin, rDNA origin) was purchased from Eli Lilly (Toronto, ON, Canada).