Fig. 3. The PTB domain of Mint3 is functionally required for localization of Furin in the TGN. (A) Schematic representation of Mint3 and its various deletion mutants. The binding abilities of His-tagged fusion proteins to Furin are summarized to the right based on the pull-down assays shown in B. (B) Mint3 interacts with Furin through its PTB domain. His-tagged fusion proteins were immobilized on TALON metal affinity beads and incubated with HeLa cell lysates. The upper panel shows a specific immunoblot for Furin from the pull-down lysates, and the lower panel represents the Coomassie staining of His-tagged fusion proteins in the pull-down assays. TCL (total cell lysate) is shown as 1% of input. (C) The PTB domain of Mint3 is necessary for the localization of Furin at the TGN. HeLa cells were co-transfected with pCMV-Furin, human Mint3 shRNA plasmids and with the GFP-tagged mouse Mint3 constructs indicated in the left panel. Cells were fixed and immunostained with antibodies to Furin and Mint3 as indicated. Scale bars: 5 µm.