Fig 2.

(A) (Left) Time-lapse confocal microscopy. bEnd.3 monolayers expressing GFP–JAM-A were exposed to CCL2 (100 ng/ml). Images were obtained every 5 min from 0 to 120 min. Representative images are from some critical time points during CCL2-induced alterations in brain endothelial cell barrier permeability. There is a fragmented pattern of JAM-A staining at the cell-cell border of endothelial cells during CCL2 exposure. (Right) TEER after exposure (0 to 120 min) to CCL2 or LPS. Data represent averages ± SDs for 5 independent experiments. (B) Triple-label immunostaining with CellLight membrane-CFP Bacmam 2.0 (blue), anti-JAM-A antibody (green), and anti-ZO-1 antibody (red) supported by optical z-stack section and three-dimensional analysis clarified that after exposure to CCL2 for 30 min, JAM-A redistributed onto the brain endothelial cell apical surface, where it may play a role as a leukocyte adhesion molecule. Bar, 50 μm. (C) Cell-based ELISA of JAM-A surface expression upon exposure to CCL2 (100 ng/ml) or LPS (5 μg/ml). Cells were treated for different time points (0 to 60 min), and every sample was then incubated with anti-JAM-A antibody, followed by HRP-conjugated secondary antibody and fixation. Notice that over a short time (10 to 20 min) JAM-A disappears from the cell surface, with return and expression on the apical membrane from 30 to 60 min. Data represent averages ± SDs for 3 independent experiments. *, P < 0.05 compared with control nontreated cells; **, P < 0.01 compared with control nontreated cells; ***, P < 0.001 compared with control nontreated cells. ODS 450, optical density at 450 nm.