Dock180 interacts with GRASP in live cells. A split YFP system was used to demonstrate the interaction of Dock180 and GRASP in cells. Dock180 and GRASP were respectively labeled with the N and C-terminal halves of venus (VN-Dock180 or VC-GRASP). Neither VN nor VC is fluorescent by itself. YFP fluorescence is reconstituted if the two fusion partners interact, thereby bringing VN and VC into close proximity so that a functional YFP can be formed. MDCK cells were transfected with the indicated constructs using Neon® (1400, 20, 2). They were then plated on Fibronectin coated coverslips. After 18 hours, Cells were fixed and stained with mouse anti-myc (cytohesin 2) or mouse anti- Flag (Dock180) followed by Dylight™ 594- conjugated anti-mouse secondary antibody and rabbit anti-HA (GRASP) followed by Dylight™649- conjugated anti-rabbit secondary antibody. In the merge cytohesin 2 or Dock180 are psuedocolored red, GRASP is psuedocolored blue, and the YFP channel detecting Venus indicating that the 2 expressed proteins interact is pseudocolored green. Bar, 10 μm.
Attar and Santy BMC Cell Biology 2013 14:9 doi:10.1186/1471-2121-14-9