AT inhibits GAG-dependent HRPII-mediated inflammatory responses in endothelial cells. (A) Confluent endothelial cells (hTERT-HUVECs) were simultaneously treated with HRPII (100 nM) and AT (2.5 µM) for 15 min followed by analysis of phosphorylation of NF-κB p65 by western blotting. Quantitative analysis is presented. (B) Cells were treated similarly with HRPII (100 nM) and AT or AT-4Mut (2.5 µM) for 1 hour and then fixed, permeabilized, and stained with anti–NF-κB p65 antibody (rabbit) followed by Alexa Fluor 488-conjugated anti-rabbit immunoglobulin G (IgG). DAPI was used to stain the nucleus. (C) Cells were treated with HRPII and AT derivatives as described above for 4 hours followed by analysis of cell surface expression of ICAM-1 by flow cytometry. Quantitative analysis is presented. (D) FITC-conjugated HRPII (HRPII-FITC, 1 µM) binding to endothelial cells were analyzed by flow cytometry in the absence and presence of the GAG antagonist, surfen (10 µM). Quantitative analysis is presented. (E) Cells were treated with HRPII (100 nM) for 1 hour in the absence and presence of surfen (10 µM). Cells were then fixed, permeabilized, and stained with anti–NF-κB p65 (rabbit) antibody and Alexa Fluor 488-conjugated anti-rabbit IgG. DAPI was used to stain the nucleus. (F) Confluent cells were treated with HRPII in absence or presence of surfen (10 µM) followed by monitoring HRPII-mediated barrier-disruptive function through measuring the influx of albumin-bound Evans blue dye across the cell monolayer as described in "Materials and methods." (G) The same as (F) except that the competitive effect of increasing concentration of wild-type AT and AT-N135Q were monitored in presence of fixed concentration of HRPII (40 nM).