Figure 1.

Kinetics of TNF expression in human monocytes and macrophages post LPS treatment. Human CD14+ monocytes (A) or day 7 human CD14+ monocyte-derived macrophages (B) were either untreated or treated with LPS in the presence or absence of 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. Na, K-ATPase and GAPDH protein expressions were used as total protein loading and cell surface biotinylation controls. Cell-free culture supernatants from monocytes (C) and macrophages (D) as treated in (A) and (B), respectively, were assayed for the presence of soluble TNF (sTNF) by ELISA.