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	Figure 1. Selectivity properties of hydrogels derived from Xenopus FG domains. (A) Macroscopic pictures of Nup98 (wild type; wt), Nup98 (NQ⇒S mutant) and Nup358 FG hydrogels. The used Nup358 domain combines all nine FG subdomains of the protein (Table I). Gels were formed on parafilm, inverted and photographed. (B) Simultaneous influx of mCherry and NTF2-Al488 into a Nup98 FG (wt) hydrogel measured after 30 min by confocal laser-scanning microscopy. The far-red labelled hydrogel was detected after excitation at 633 nm, mCherry at 561 nm and Alexa488-labelled NTF2 at 488 nm. Arrow illustrates direction of influx. Concentration profiles of mCherry and NTF2 across the buffer/gel boundary are also shown. For normalization, free NTF2 and mCherry concentrations in the buffer had been set to 1. (C) Two-hour-influx of NTF2, mCherry and tCherry into 10 different Xenopus FG hydrogels (for sequences of used domains, see Supplementary Figure S1 and Supplementary Table S1). False-coloured fluorescent signals illustrate partitioning of mobile species between buffer and gels. Note that the isolated FG domains of Nup54 and Nup58 show non-specific binding to tCherry (see also Figure 4).
	Figure 2. Selectivity properties of Nup214 hydrogels. (A) Domain organization of Xenopus laevis Nup214. Red strokes represent FG motifs. Nup214 contains a canonical FG domain (‘FG') as well as two FG-like domains. (B) Photographs of indicated hydrogels. (C) Simultaneous influx of NTF2 and mCherry or of Impβ·IBB-GFP and tCherry into indicated hydrogels (2 h time points). Permeation assays were performed as in Figure 1. Note the deviating scaling of NTF2 and Impβ·IBB-GFP signals for the FG-like hydrogels. (D) Influx of RanGTP·GFP-CRM1·NES export complex into Nup214 hydrogels as compared to Nup358 FG hydrogels (6 h time points). Influx of free GFP-CRM1 and of a CAS export complex is shown as controls.
	Figure 3. FG subdomains of Xenopus tropicalis Nup153 form highly selective hydrogels. (A) Photographs of indicated gels. (B) Permeability properties of Nup153-derived FG hydrogels were analysed as in Figure 2C (2 h time points).
	Figure 4. Permeability properties of hydrogels derived from the Xenopus laevis Nup62 complex. (A) Domain organizations of Nup54, Nup58 and Nup62. (B) Hydrogels obtained from indicated nucleoporin fragments or domain fusions were probed with NTF2, mCherry or tCherry as in Figure 1B (30 min time points). Please note that the Nup54 and Nup58 hydrogels here also include the FG-like domains, which had been omitted in Figure 1C.
	Figure 5. Analysis of Nup98 FG hydrogels. (A) The non-glycosylated Nup98 FG domain forms an inhomogeneous hydrogel at the concentrations below 200 mg/ml. Microscopic pictures illustrate the appearance of saturated and non-saturated Nup98 FG hydrogels. (B) Potential O-GlcNAc modification sites within the Nup98 FG domain were identified by mass spectrometry after enzymatic in vitro glycosylation. Modified Ser and Thr residues are shown in red, non-sequenced regions as strikethrough text, FG motifs in bold, the GLEBS domain is underlined. (C) Non-glycosylated or O-GlcNAc-modified Nup98 FG hydrogels were probed with indicated mobile species as described in Figure 1C (2 h time points). (D) The non-glycosylated and O-GlcNAc-modified Nup98 FG domains, as well as the Nup98 FG (Ф⇒S) mutant were immobilized on Ni(II) Silica beads and incubated with a pre-formed Impβ·IBB-mCherry complex. Bound fractions were analysed by SDS–PAGE and Coomassie staining. The Ф⇒S mutant, where all hydrophobic residues had been exchanged to serines, runs slower than expected from its mass.
	Figure 6. WGA inhibits passive diffusion and facilitated entry into an O-GlcNAc-modified Nup98 FG hydrogel. The gels were pre-incubated with either buffer (left column) or WGA and subsequently probed with mCherry, NTF2 and an Impβ·cargo complex (30 min time points).
	Figure 7. Solid-state NMR analysis of scNsp1- and Nup98-derived FG hydrogels. (A) 1D cross-polarization (CP) spectra probing very rigid parts of the scNsp1 FG hydrogel (black curve), a non-glycosylated Nup98 FG hydrogel (blue) and a O-GlcNAc-modified Nup98 gel (red). (B) INEPT spectra probing highly mobile regions of the same gels (colour coding as in A). (C) 2D CP spectra probing very rigid parts of the scNsp1 gel (black) and the non-glycosylated Nup98 FG hydrogel (blue). Indicated correlations reflect inter-atomic polarization transfer among aliphatic carbon positions (denoted by α-γ) for selected residue types. Resonance frequencies encode information about the identity of residues and their backbone conformations. (D) 2D direct excitation spectra comparing non-glycosylated (blue) and O-GlcNAc-modified (red) Nup98 FG hydrogels. Resonance peaks indicating β-strand conformation are labelled in green, random coil conformations are coloured in mustard yellow.

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