Additional cargo controls. (A and B) Facilitated transport of Traptavidin-GFP-NLS in the presence and absence of Importin β/Importin α into the FG-Nup49 droplets studied using a 10× objective and epifluorescence microcopy (whole device). This experiment was particularly challenging because of precipitation of cargo/NTRs complexes inside the device (likely due to the tendency of traptavidin to multimerize). Despite this issue, we could observe a remarkable difference between the experiments in the presence and absence of NTRs (A and B, respectively). The maximum-intensity projection in the figure shows the enrichment of the cargo into the droplets along the device. As for all the facilitated transport experiments shown in this article, the functionality of this cargo was also tested in cells with and without NTRs (top row A and B, respectively). Scale bar is 50 µm. (C) Facilitated transport of IBB-mCherry in the presence of Importin β inside the FG-Nup49 droplets studied on a confocal microscope. This is the positive control corresponding to Fig. 3 C. The IBB-mCherry/Importin β mix was preincubated at a 1:1 ratio for 30 min before the start of the microfluidic experiment. The interaction between this cargo and the FG-Nup49 droplets is evident in the cargo channel. The top row shows functionality of the IBB-mCherry cargo in permeabilized cell assays. The last row shows the region of the device in which confocal imaging was performed; all images in Fig. 3 were recorded in this same region. Scale bar is 20 µm, acquired on a custom confocal microscope with a 60× objective (water).