SEPT2 fibers localize to TGN sites of apical and basolateral protein export. (A and B) Subconfluent monolayers of MDCK cells were stained with SEPT2 and p115 antibodies, and imaged by confocal microscopy. Optical sections from the bottom, middle, and top of the highlighted Golgi region are shown at higher magnification (B). (C and D) MDCK-SEPT2-YFP cells were transiently transfected with gpi-CFP or GalTase-CFP, and imaged by time-lapse microscopy at 37°C. Non-neighbor deconvolution was applied and linear dynamic ranges were renormalized equally across the entire image to enhance the fluorescence intensity of tubular–vesicular structures. Arrows point to tubular vesicular elements extending from the Golgi complex. (E) Tubular–vesicular carriers containing CFP-tagged gpi, GalTase, VSV-G, and p75 were scored for overlap with SEPT2-YFP fibers (n = 80–100; mean values ± SEM from three independent experiments). (F) 3D-rendered confocal images of a Golgi region from MDCK-SEPT2-YFP cells stained with α-tubulin and furin convertase antibodies. (G) HeLa cells were transfected with the protein kinase D1 mutant PKD-K618N (PKD1-KD) tagged with GST. Cells were stained with GST and SEPT2 antibodies. Arrowheads point to a PKD-K618N–containing tubular extension. Bars, ∼5 μm.