Characterization of the soluble peptide CD9-LEL-GST and its mutants. (A) LEL-GST fusion proteins of human CD9, as well as point mutations to Ala of Cys 152, 153, 167, and 181 were generated, produced in bacterial cultures, and isolated by affinity columns of Glutathione-Sepharose. Eluted purified proteins were then analyzed by Ponceau staining and Western blot against CD9 (VJ1/20 mAb) or GST. (B) Flow cytometry analysis of the expression of ICAM-1 and VCAM-1 in HUVECs preincubated with CD9-GST or its point mutants. Thin lines in the upper panels correspond to the expression of resting cells. Thin lines on the following panels correspond to cells treated with TNF-α alone. Thick lines correspond to the expression in cells treated for 20 hours with TNF-α alone (top row) or in combination with the different soluble LEL-GST peptides. (C) Propidium iodide profiles of cells treated for 20 hours with TNF-α alone or in combination with the different soluble LEL-GST peptides. (D) Paracellular permeability analysis of HUVEC monolayers preincubated with the different soluble LEL-GST peptides. Thrombin was added at 0.1 U/mL at the time of addition of the fluorescent dextran. Data represent the mean ±SD of a representative experiment performed in duplicate.