Figure 9.

Entry of NTR·cargo complexes into an FG hydrogel is reversible. (A) A saturated FG/FxFG_2–601^Nsp1 hydrogel was preloaded overnight with an NTR·cargo complex (1 μM of GFP-scImpβ·IBB-MBP-mCherry in the buffer). The free complex was removed by several buffer changes and phenyl–sepharose beads were placed in front of the gel. Then, confocal scans detecting cargo and receptor were started. The beads served as a trap for scImpβ and strongly accumulated NTR and cargo over time. The accumulated signal was strongest on the side that faced the gel, identifying the hydrogel as the source of the NTR·cargo complex. Bar diagrams translate false colour look-up tables into grey scale. (B) An FG hydrogel was loaded with 1 μM of NTR·cargo complex as in panel A. The first scan (1) shows the loading of the gel. (2) A preloaded gel immediately after removing the free NTR·cargo complex by three buffer changes. (3) A preloaded gel after 3 h incubation with buffer and (4) after incubation with 4 μM of GTP-Gsp1p (S. cerevisiae Ran). Gsp1p increased the efflux of the cargo and of scImpβ from the gel. Two different scan settings of the experiment are shown, optimised to visualise either low or high protein concentrations.