Figure 2.

Kinetics of TNF expression in monocyte-derived dendritic cells post LPS treatment. Day 5 human CD14+ monocyte-derived DCs were either untreated or treated with LPS in the absence (A) or presence (B) of 20 μM TACE inhibitor, TAPI-2, for the indicated periods of time. Cell surface proteins were labeled with cell-impermeable Sulfo-NHS-SS-biotin. Biotinylated surface proteins were precipitated with streptavidin-conjugated agarose beads. Cell surface biotinylated and total proteins were subjected to immunoblotting using anti-TNF IgG. Cadherins and GAPDH protein expressions were used as total protein and cell surface biotinylation loading controls. Cell-free culture supernatants of DCs as treated in (A) and (B) above were assayed for the presence of soluble TNF (sTNF) (C) by ELISA.