Figure 7
From: A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association
Visualization of complex formation in mammalian cells.
(a) Leucine zipper and Ku70/80 heterodimerization. Cells transiently expressing GFP1–9 with GCN4 zipper (Z-11) (left panel) or Ku70-GFP11 (Ku70-11) (right panel) display no background fluorescence. Heterodimerization is visualized as fluorescence with interacting leucine zippers GFP10-Z and Z-GFP11 in CHO cells expressing GFP1–9. Complementation of GFP11-Z with GFP1–10 confirms localization of the zipper alone (bottom). Ku70/80 complex formation is visualized in cell nuclei (right panel). Expression of one Ku component tagged with GFP11 is monitored with GFP1–10. FACS analysis of HEK 293_GFP 1–9 cell lines transfected with corresponding constructs (24 h after transfection). Percentage of fluorescent cells (black bars); mean fluorescence intensity of the positive cell population (gray bars). Self-associating GFP10-Z-GFP11 domain is used as positive control of transfection and complementation with GFP1–9 (mean ± SD; N = 3). (b) Rapamycin induced FRB/FKBP interaction in mammalian cells. Stable HEK 293 cells expressing GFP1–9 were co-transfected with GFP10-FRB and FKBP-GFP11 constructs and stimulated with increasing concentrations of rapamycin (RAP) (0, 10, 20, 50 and 100 nM). Bipartite complementation of FKBP-11 and GFP1–10 is shown in the most left image; Green fluorescence at 488 nm excitation (GFP), DAPI nuclear staining (cyan). Scale bars = 10 μm. Bottom panel: FACS quantification of rapamycin induced association with or without addition of competitive inhibitor FK-506 (1 μM). Right graph: addition of increasing concentrations of FK-506 (20 nM rapamycin; 0.01, 0.1 and 1 μM FK-506) (mean ± SD; N = 3).
