Perk1 2 9101

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.

Cortical kidney lysates with equal amounts of total protein (50 μg/ml) were fractionated by 7.5–15% SDS-polyacrylamide gels under reducing conditions and analyzed by immunoblot. The antibodies against fibronectin, collagen I (ab34710, Abcam), MR, arginase I (ab64693, Abcam), NGAL, KIM-1, TNF-α, p38 (sc-535, Santa Cruz Biotechnology), MCP-1, phosphorylated-p38 (p-p38, 9211, Cell Signaling Technology, Danvers, MA, USA), ERK1/2 (sc-292838, Santa Cruz Biotechnology), GSK3β (sc-53931, Santa Cruz Biotechnology), phosphorylated GSK 3β-Ser 9 (p-GSK3β-Ser 9, sc-11757, Santa Cruz Biotechnology), phosphorylated ERK (p-ERK 1/2, 9101, Cell Signaling Technology), JNK (sc-572, Santa Cruz Biotechnology), phosphorylated JNK (p-JNK, ab124956, Abcam) and GAPDH (sc-48166, Santa Cruz Biotechnology) were used as primary antibodies.

The ADAM8 antibody (B4068) used for Western blotting was purchased from LifeSpan Biosciences. Antibodies against β-actin (AC-15) and β-tubulin (TUB 2.1), and the ERK inhibitor FR180204 (SML0320) were obtained from Sigma-Aldrich. The antibody to detect specific ERK 1/2 phosphorylated forms (pERK1/2) (9101) was obtained from Cell Signaling Technology. The β1-integrin antibody (552828) and its control isotype matched IgG2A (SC3883) were purchased from BD Biosciences and Santa Cruz Biotechnology, respectively. The anti-ADAM8 antibody MAB10311 and its control isotype-matched IgG1 (MAB002) were from R&D Systems.

CProteins were extracted in RIPA buffer supplemented with phosphatase and proteinase inhibitors. Protein concentration was determined with the Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Details on Western blotting procedures were previously described [34 (link)]. In brief, equal quantities of protein were resolved using SDS-PAGE and transferred onto nitrocellulose membranes, blocked in 5% dry fat milk, incubated with primary antibodies at 4°C and subsequently with corresponding secondary antibodies at room temperature. Developed membranes were imaged with a ChemiDoc MP Imaging System (BioRad, Hercules, CA, USA) and quantified with ImageLab software (BioRad, version 5.2.1). The following primary antibodies were used (diluted 1:1’000 except Actin, which was diluted 1:5’000): pERK1/2, #9101; p-p38, #9212, pSTAT3, #9145; ATGL, #2138; pHSL #4139 (all from Cell Signaling); Gapdh, G9545 (Sigma-Aldrich), Actin, MAB1501 (Millipore), GLUT1 and GLUT4 (gift from Dr. A. Klip, The Hospital for Sick Children, Toronto, ON, Canada; samples were not boiled for Western blots assessing GLUT1 and GLUT4 protein levels).