Figure 4.

Facilitated translocation tightens the FG/FxFG hydrogel even against passive influx of GFP-sized objects. The influx experiment was carried out as in Figure 3, but two differences were implemented: First, we used a smaller inert reference molecule (mCherry, Stokes radius (R_S)=2.4 nm) in order to enhance the sensitivity for small changes in passive permeability. Second, for pre-incubation, the ‘empty' scImpβ was replaced by an IBB-MBP-mEGFP·scImpβ complex. The receptor used for the pre-incubation was therefore also detectable in the GFP channel. As expected from its smaller size, mCherry entered the untreated gel considerably faster than the MBP-mCherry fusion. The pre-incubation with the NTR·cargo complex diminished mCherry influx to very low levels. The pretreatment did, however, not abolish the NTR-mediated cargo entry (see Supplementary Figure 1).