Chemidoc touch station

HRMECs or tissues were lysed by lysis buffer (Cell Signaling, Topsfield, MA, USA). Total protein (50 μg) was loaded on Bio-Rad 4–20% gel system, transferred to PVDF membrane, and immunoblotted with one of the following primary antibodies: Serine473 phosphorylated Akt (Cat#4060, Cell Signaling), total Akt (Cat#4691,Cell Signaling), Threonine172 phosphorylated AMPK (Cat#2535, Cell Signaling), total AMPK (Cat#5832, Cell Signaling), Acetyl-CoA Carboxylase (Cat #3662, Cell Signaling), Phospho-Acetyl-CoA Carboxylase (Cat #11818, Cell Signaling), Claudin-1 (Cat#13995, Cell Signaling), zo-1 (Cat#13663, Cell Signaling), Claudin-5 (Cat#35-2500, Thermo-Fisher Scientific), Occludin (Cat#71-1500, Thermo-Fisher Scientific), and ß-Actin (Cat#SC-4778, Sigma). The secondary antibody was obtained from Cell Signaling Company. The membrane was exposed to Super-Signal Reagent (Pierce, IL, USA), and imaged on a Bio-Rad ChemiDoc Touch station (Bio-Rad).

HRMECs and mice retinal tissues were lysed using lysis buffer (Cat# 9803, Cell Signaling Technology, Topsfield, MA, USA). The total protein (50 μg) was loaded to the Bio-Rad gel (4–20%) and subjected to electrophoresis, then was transferred to a polyvinylidene fluoride membrane, and immunoblotted with primary antibodies against serine 473 of phosphorylated Akt (Cat# 9271), total Akt (Cat#4691), phosphorylated ERK (Thr202/Tyr204) (Cat# 9106), total ERK (Cat# 9107), heme oxygenase 1 (HO-1; Cat# 70081), peroxiredoxin 1 (Prdx-1; Cat# 8499), cleaved caspases-3 (Cat# 9661), total caspase-3 (Cat# 9662), β-actin (Cat#SC-4970), and the secondary antibodies (Cat#58802 and Cat#91196, all antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA). After being treated with Super-Signal reagent (Cat#34095, Pierce, IL, USA), the membrane was photographed using a Bio-Rad ChemiDoc Touch station (Bio-Rad Laboratories, Hercules, CA, USA).