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. Author manuscript; available in PMC: 2016 Aug 15.
Published in final edited form as: Nat Genet. 2016 Feb 15;48(4):457–465. doi: 10.1038/ng.3512

Figure 3. NUP93 mutations interfere with nuclear pore complex (NPC) assembly, and a NUP205 mutation affects NUP93-NUP205 interaction.

Figure 3

(a) Upon overexpression in human podocytes, Myc tagged NUP93 but not the splice site mutation del ex13 or the truncating mutation Lys442Asnfs*14, localize to the nuclear envelope (arrow heads). Scale bar 10 μm, see also c. (b) Mutations identified in individuals with SRNS fail to assemble an intact NPC in a depletion-addback assay in X. laevis egg extracts. Upon depletion of NUP93 nuclei fail to assemble properly. Addback of full-length or the mutant constructs Gly591Val or Tyr629Cys restore nuclear envelope and NPC assembly. The constructs of the in-frame deletion of exon 13 (del ex13), the missense mutation Arg388Trp, and the frameshift mutation Lys442Asnfs*14 lack this ability. Nuclear membranes are stained with DiIC18 (red, upper row), DNA with DAPI (blue), NPCs with mAB414 (second row, red), and α-Nup93 antibody (third row, green). Scale bar 10 μm, see also d. (c) Quantitation of nuclear localization data from (a) resulting from 50 transfected cells for each condition. (d) Quantitation of depletion-addback assay data from (b) presented as mean and standard deviation resulting from 100 nuclei each from three independent experiments. (e) Upon shRNA knockdown of NUP93 (left panel) immortalized human podocytes show a reduced expression level of NUP205 (right panel). (f) GFP tagged NUP205 precipitates endogenous NUP93 upon overexpression in HEK293 cells. A mutant allele of NUP205 identified in SRNS family A1733 (Phe1995Ser) lacks this interaction.