Interactions of NXF1 with Nups in single NPCs detected using FLIM-FRET. (A–C) Immunostaining and LT analysis of NXF1 (donor) along with (A) Nup358, (B) Nup214, and (C) Tpr (acceptor). Cells were immunostained with either donor-only or donor + acceptor primary antibodies and with two secondary antibodies (Alexa Fluor 488 donor and Alexa Fluor 647 acceptor). Histograms show the LT values of the NXF1-donor in single NPCs in donor-only cells (blue) compared with donor + acceptor cells (red), in a representative experiment. Box plots of the LT values show the reduction in lifetimes when the Nup358 or Nup214 acceptor are introduced. Light lines represent data from single cells, and the bold plot contains data from all cells. ns, nanoseconds. See Materials and methods for the number of NPCs and cells shown. ***, P < 0.001; *, P < 0.05. Scale bars, 10 µm. (D) Summary of the FLIM-FRET measurements between NXF1 and indicated Nups (see Fig. S3). Dark blue indicates Nups that were found to interact with NXF1.