Western blot analysis of sTRAIL released by IFNα-stimulated neutrophils and PBMCs. Supernatants harvested from neutrophils and PBMCs, cultured for 24 hours with 100 U/mL IFNα, were concentrated and analyzed for the presence of sTRAIL by Western blot, as described in “Patients, materials, and methods.” Data are representative of analyses performed in 2 healthy donors. (A) Concentrated supernatants (50 μg/sample) from neutrophils (lane 1) and PBMC (lane 2) were run under reducing conditions and stained with anti-TRAIL mAb. The rhsTRAIL (nondisulfide-linked homotrimer) at 100 ng/sample (lane 3) was included as positive control. (B) The rhsTRAIL (lane 1), supernatants of activated neutrophils (lane 2), and PBMCs (lane 3) were analyzed by Western blot under nonreducing conditions; samples were heated at 60° C for 15 minutes before loading. (C) Freshly concentrated supernatants of activated PBMCs (lanes 1-2) and neutrophils (lane 3-4) were analyzed under native conditions in comparison with rhsTRAIL (lane 5).