Figure 7.

A1 protein expression in human primary neutrophils upon pro-inflammatory stimulation and inhibitor treatment. (a) Immunoblot analysis of human peripheral blood-derived primary neutrophils. Human neutrophils were isolated from peripheral blood of two different healthy donors by Histopaque/Percoll density centrifugation and were stimulated for 20 h with human recombinant GM-CSF (10 ng/ml) or LPS (1 μg/ml) where indicated. Cells were additionally treated with LY (20 μM), JI1 (1 μM) or U0126 (10 μM) or solvent control (0.5% DMSO). Proteins were extracted by lysis and boiling in Laemmli buffer. Whole-cell lysates (equivalents 1 × 10⁶ cells per lane) were separated by SDS-PAGE, transferred onto a nitrocellulose membrane and probed for Bfl-1/A1 and Mcl-1. β-Actin served as a loading control. Shown are blots derived from cells of two healthy individuals. (b) Survival of human primary neutrophils during pro-inflammatory stimulation in the presence or absence of various kinase inhibitors. Cells isolated from human healthy donors as described in a were stimulated with human recombinant GM-CSF or not in the presence of kinase inhibitors or solvent control as indicated above for 20 h. Apoptosis was analyzed by Annexin V/PI staining followed by flow cytometry. Data show survival as determined by the percentage of Annexin V/PI-negative cells. Bars represent mean/S.E.M. of two experiments with cells from two donors each (n=4 in total)