IL-6 exits the TGN in carriers both with and without TNFα. (A and B) Live epifluorescence dual-color imaging of macrophages transfected with TNFα-mCherry (red) and IL-6–GFP (green) demonstrating IL-6 leaving the TGN in a carrier devoid of TNFα (A) and in a tubule also containing TNFα (B). Cells were stimulated with LPS and IFNγ before imaging. In the enlarged images, arrows are used to highlight single trafficking events, and the time at which each still frame was collected is labeled. (C) Cell lysates of activated macrophages were fractionated to separate Golgi membranes and cytosol. Golgi membranes were then incubated with the cytosol in an in vitro Golgi budding reaction as described in Materials and methods. Samples from the budded vesicle fractions (labeled 1–9) were resolved and analyzed by Western blotting to detect γ-adaptin as a vesicle-associated protein and IL-6 and TNF as cargoes. Bar, 10 μm.