Fig. 4. N4WBP5A localizes to multivesicular bodies and Golgi vesicles. HeLa cells transiently transfected with N4WBP5A-myc were fixed with paraformaldehyde and processed for cryoelectronmicroscopy. Ultrathin sections were double labelled with monoclonal anti-myc antibody and Nedd4 (A,B) using 10 nm or 15 nm goat anti-mouse or goat anti-rabbit gold conjugates. (A) Localization of N4WBP5A-myc (arrows) in the TGN and Nedd4 (arrowheads) in the TGN (G, TGN; pm, plasma membrane). (B) Localization of N4WBP5A-myc and Nedd4 (arrowheads) in MVBs. (C) Localization of N4WBP5A-myc in a MVB showing abundant labelling on the internal vesicles and some labelling on the limiting membrane. Bar, 200 nm. (D) Western blot analysis of N4WBP5 and N4WBP5A in Golgi-derived vesicles. Golgi membranes (G) were incubated with rat liver cytosol (C), GTPS and HKM buffer. A mixture of rat liver cytosol and cytosol from transfected cells was used to assay the binding of recombinant proteins to the Golgi membranes. After budding, the remnant Golgi cisternae (Gb) were separated by centrifugation, and the Golgi-derived vesicles (V) were separated from the remaining cytosol (C2). The vesicle pellet was prepared on a 20-50% discontinuous sucrose gradient and fractions (1-9) collected and analysed by western blot using appropriate antibodies.