FIGURE 3.

CD14 is present on the cell surface and released as a soluble variant in itraconazole-treated macrophages. a, RAW 264.7 Mϕs were treated with itraconazole (1 μm) or the respective carrier (DMSO) for 16 h followed by biotinylation and purification of cell surface proteins (Pierce Cell Surface Biotinylation kit). Fractions of both total lysates and cell surface proteins were analyzed by Western blot for CD14. Bands were quantitated by densitometry and % cell surface CD14 was calculated. b, RAW 264.7 Mϕs were treated with itraconazole (1 μm) or the respective carrier (DMSO) for 16 h followed by stimulation with 100 ng/ml LPS for additional amounts of time. Both lysates and supernatants were collected and CD14 was analyzed by Western blot.