Anti phosphorylated p jnk

The BMSCs transfected with miR-214 or anti-miR-214 plasmids, or treated with JNK inhibitor or p38 inhibitor were lysed on ice for 30 min in RIPA lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitor. The protein concentration was determined by Bradford assay (Bio-Rad) on a microplate spectrophotometer (Tecan). A total of 40 µg proteins was resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Pall Corporation, New York, NY, USA). The membranes were incubated with the appropriate primary antibodies [anti-fibroblast growth factor (FGF; 1:1,000; 9740), anti-phosphorylated (p-)JNK (1:2,000; 4668), anti-p-p38 (1:2,000; 4511) (all from Cell Signaling Technology, Danvers, MA, USA) and GAPDH (1:5,000; H00002597-D01P; Abnova, Taiwan, China) at 4°C overnight. The membranes were then incubated with an HRP-conjugated secondary antibody (Xi'an Kehao Biological Engineering Co. Ltd, Xi'an, China) and developed by enhanced chemiluminescence (ECL; Millipore, Billerica, MA, USA). Protein expression was analyzed using an Odyssey Two-Color Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

For western blot, 30 μg of retinal proteins were loaded in each lane and separated by electrophoresis on 12% (v/v) sodium dodecyl sulphate-polyacrylamide gels, and then transferred to polyvinylidene difluoride membranes (Millipore). Membranes were incubated with PBS containing 5% dried non-fat milk for 1.5 hours at room temperature and then probed with primary antibodies including rabbit anti-JNK (1:1000, Cat# 9252), anti-phosphorylated (p-) JNK (1:1000, Cat# 4668), anti-extracellular-signal-regulated kinase (ERK; 1:1000, Cat# 9102), anti-p-ERK (1:2000, Cat# 4370), and anti-glyceraldehyde 3-phosphate dehydrogenase (1:5000, Cat# 2118; all from Cell Signaling Technologies, Danvers, MA, USA) at 4°C overnight. After incubation with goat anti-rabbit horseradish peroxidase-conjugated IgG (1:5000, Cat# 111-035-003, Jackson ImmunoResearch, West Grove, PA, USA) for 1.5 hours at room temperature, blots were washed three times with PBS and incubated with an enhanced chemiluminescence reagent (Bio-Rad Laboratories). Image documentation, processing, and quantification were similarly performed with ImageJ software as mentioned above. A ratio for the total intensity of the target protein over that of the loading control was calculated and normalized to WT group. Measurements were repeated for at least 3 times in each experiment.