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Fig. 3. Vglut1 and ZnT3 are targeted to a common vesicle population. A glycerol gradient isolated SLMV fractions derived from Vglut1 and ZnT3HA (VglZn) double-transfected PC12 cells, and these were immunomagnetically isolated with beads coated with monoclonal antibodies against Vglut1 (A-C), antibodies against the HA tag present in ZnT3 (D, lane 2) or a monoclonal antibody against the AP-3 {delta} subunit (D, lane 3). Control assays were performed with beads coated with an antibody against a lumenal epitope of LAMP I. Bead-bound vesicles were resolved by SDS-PAGE and analysed by immunoblot with polyclonal antibodies against Vglut1, ZnT3 and the AP-3 ß3 subunit, and a monoclonal anti-transferrin-receptor (TrfR) antibody. (B) Unbound membranes were sedimented at high speed and analysed in parallel with bead-bound organelles depicted in (A,D). The Vglut1 blot was exposed for 30 seconds, whereas the TrfR blot was exposed overnight. Inputs correspond to 10%. Lanes 2 and 3 (A-C) represent duplicate assays.





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