(A) Scheme of experimental design. Incubation of human brain microvascular endothelial cells (HBMEC) with TNF-α (100 ng/mL) leads to secretion of sVEcadherin fragments into the media, which is analyzed subsequently with ELISA and Western blot. (B) Immunofluorescent staining with an antibody to the extracellular (EC) domain of vascular endothelial (VE)-cadherin. The number of gaps in the VE-cadherin signal is increased after 18 hours incubation with TNF-α (100 ng/mL) (p < 0.001 for time or treatment; 2-way analysis of variance [ANOVA], ** p < 0.01, *** p < 0.001, ## p < 0.01, ### p < 0.001; with Tukey multiple comparison, n = 4). (C) Increased sVE-cadherin is seen in Western blots of concentrated cell culture media (same conditions as in A and B) after 18 hours incubation with TNF-α (p = 0.043 with one-way ANOVA; * p < 0.05 with Dunnett multiple comparison, n = 4). IB = immunoblot.