T iκb

The retina of mice and BV-2 or HRECs were collected and homogenized with lysis buffer, followed which centrifugation (1600 × g, 15 min) was performed at 4°C. Protein concentration was quantified in supernatant fluid by bicinchoninic acid assay (Beyotime Institute of Biotechnology, Haimen, China). Separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to the PVDF membranes (GE Healthcare Europe GmbH, Freiburg, Germany). Proteins were blocked with 5% nonfat milk for 1 h and incubated with primary antibodies (p-p65: ab76302; t-p65: ab32536; occludin: ab216327; claudin-1: ab180158; ZO-1: ab216880; VEGF: ab214424; CD31: ab182981; GAPDH: ab8245. Abcam, England. p-IκB: #2859, t-IκB: #9242, Iba-1: #17,198, Cell Signaling Technology, USA). Subsequently, HRP-conjugated secondary antibody (ab7090, abcam, England) was used to incubate the membranes for 1 h at 37°C. Protein bands were visualized by enhanced chemiluminescence reagent (Millipore Corp., Bedford, MA).

Streptomycin (100 ng/mL) and penicillin (100 U/mL) were purchased from Sigma (Gillingham, UK); 96 well plates were provided by NUNC, (Havel, Germany), while GIBCO (Grand island, NY) provided fetal bovine serum (FBS). DMSO, dexamethasone, lipopolysaccharides (LPS—Escherichia coli O111:B4), N-nitro-l-arginine methyl ester (L-NAME), Griess reagent (1% sulfanilamide/0.1% naphthyl ethylenediamine dihydrochloride in 2.5% H₃PO₄), camptothecin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were bought from Sigma, IL-6 and TNF-α ELISA Kits were procured from Invitrogen (Massachusetts, US). Synergy Mx microplate reader belonged to BioTek, Winooski, VT, USA. P-IκB, T-IκB, P-p65-NF-κB, T-p65-NF-κB, P-p38-MAPK, and T-p38-MAPK, GAPDH, and anti-rabbit IgG horseradish peroxidase (HRP) conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).