Localization of unprocessed TNF in IL-1β–stimulated WT and VAMP-8–deficient BMMCs. (A) VAMP-8–deficient and WT BMMCs were exposed to vehicle (3 hours). Cells were then stained with anti-TNF and anti–VAMP-8 or anti–VAMP-3 as indicated. (B) VAMP-8–deficient and WT BMMCs were stimulated with 25 ng/mL IL-1β (3 hours) in the presence of 50 μM TAPI, which inhibits cleavage of the membrane precursor form of TNF. Cells were stained with anti-TNF and anti–VAMP-8 or anti–VAMP-3 as indicated and were then analyzed by confocal microscopy. Representative single optical sections are shown. In the 2-color overlay image (M), compartments containing both markers appear as light blue.