Western blot analysis

24 h after restoration from OGD, bEnd3 cells were harvested and lysed with lysis buffer (1% Nonidet P-40 [NP-40], 50 mM Tris HCl, pH 8.0, 150 mM sodium chloride) supplemented with protease and phosphatase inhibitor cocktails. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Eppendorf-Bio Photometer, Germany). Western blotting and semiquantitative analyses were performed as described previously.10^,11 The following primary antibodies were purchased from Invitrogen (Carlsbad, CA, USA): Armenian hamster anti-ICAM-1 monoclonal antibody (3E2B, MA5405, 1:20), rabbit anti-NF-κB polyclonal antibody (51-3500, 1:1,000), rabbit anti-p-NF-κB polyclonal antibody (PA5-37658, 1:1,000), rabbit anti-E-selectin monoclonal antibody (15, MA5-29785, 1:1,000), rabbit anti-VCAM-1 monoclonal antibody (SA05-04, MA5-31965, 1:1,000), rabbit anti-KLF4 (PA5-27440, 1:5,000), rabbit anti-Claudin-5 polyclonal antibody (34-1600, 1:170), and rabbit anti-ZO-1 polyclonal antibody (61-7300, 1:1,000). The rabbit anti-Itgα5 polyclonal antibody (AB1928, 1:1,000) was gotten from Merck Millipore (Darmstadt, Germany), and β-actin was obtained from Neomarker (Fremont, CA, USA; 1:1,000,). Within each sample, levels of proteins were first normalized to the level of β-actin and then expressed as the fold increase over the level of the NO-OGD/R control group.