The Nuclear Translocation Assay for Intracellular Protein-Protein Interactions and its Application to the Bcr Coiled-Coil Domain

Figures & data

Figure 1. The NTA concept.

If the two proteins interact, as is the case for proteins X and Y in the top half of the diagram, protein X will cause an increase in the nuclear localization of protein Y upon addition of ligand L, due to the ligand-responsive PS fused to protein X. If two proteins do not interact, as is the case for proteins X and Z in the bottom half of the diagram, the PS translocates the fused protein X to the nucleus upon addition of ligand without altering the localization of protein Y. The translocation of the PS construct is monitored through the fluorescence of EGFP, and the translocation of the second protein is monitored through the fluorescence of DsRed.

Figure 2. PNI of PS constructs and example cell images.

(A) PNI of the PS constructs as measured by EGFP fluorescence. Consistent PNI of all PS constructs contributes to validating that the nuclear increase of the DsRed construct is indicative of the interaction. (B) Example images of 1471.1 cells cotransfected with EGFP-PS-CCmut and DsRed-CC. The phase contrast is seen in the top row, the fluorescence from EGFP in the middle row, and fluorescence from DsRed in the bottom row. The left column contains cells without ligand added, and the right column contains cells 2 h after ligand was added. For the PS construct (EGFP), the initial localization is in the cytoplasm, and after addition of ligand, it translocates into the nucleus. The nuclear translocation of the DsRed construct is not as noticeable, but also moves into the nucleus after addition of ligand due to the interaction with the PS construct.

Figure 3. NTA results for the Bcr coiled coil and mutant coiled coil.

The mutant coiled coil is designed to prevent homo-oligomerization (CCmut:CCmut) and to improve binding to the wild-type coiled coil. This results in EGFP-PSCCmut being more available to oligomerize with DsRed-CC and the greatest nuclear translocation with this combination (CCmut:CC, third column). PS, protein switch; CC, coiled coil; CCmut, mutant coiled coil. *P < 0.01, **P < 0.001 compared with control (first column); each experiment was performed in triplicate with at least eight cells analyzed per experiment. Statistical significance was determined using one-way ANOVA with Tukey's post-test.