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. 2023 May 8;222(6):e202210059. doi: 10.1083/jcb.202210059

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© 2023 Amm et al.

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Figure S1. — Chaetomium Nup120 and Nup133 interact with Ndc1. (A) CtNdc1 used for reconstitution in GUVs was Ni-NTA-purified from E. coli BL21 (DE3). CtNdc1 was additionally N-terminally fused to MISTIC, a B. subtilis protein allowing high-level expression. Elution was done by imidazole treatment. Before GUV reconstitution, the MISTIC tag was cleaved off using thrombin protease. The different fractions obtained during purification were analyzed via SDS-PAGE and Coomassie staining. (B) ProtA-tagged CtNup85, CtNup120, and CtNup133 were expressed in S. cerevisiae under the control of the GAL1-10 promoter and purified using IgG beads. Purified proteins were cleaved off the beads using TEV protease. Elution fractions were subjected to SDS-PAGE and Coomassie staining. (C) SCL1/BC08 was expressed and purified from E. coli BL21 (DE3), as described for CtNdc1, Alexa Fluor 488-labeled, and reconstituted into GUVs. SCL1/BC08-GUVs were incubated with either Alexa Fluor 546-labeled CtNup120 or CtNup133. Binding was monitored as described in Fig. 1. Bars, 10 µm. (D) Expression and purification of CtNup120βM consisting of an α-helical extension of the β-propeller domain of CtNup120 and CtNup133α6, an alpha-helical stretch in the C-terminal region of CtNup133. Elution fractions were subjected to SDS-PAGE and Coomassie staining. (E) SCL1/BC08-GUVs were incubated with either Alexa 546-labeled Nup120βM or Nup133α6. Binding was monitored as described earlier. Bars, 10 µm. (F) Expression and purification of CtNup120β, CtNup120βM, and CtNup133α6. (G) CtNdc1-GUVs were incubated either with Alexa Fluor 546-labeled CtNup120β, CtNup120βM or CtNup133α6. Bars, 10 µm. (H) (I) N- (1-308) or (II) C-terminal (242-646) fragments of CtNdc1, expressed, purified and labeled as above, were reconstituted into GUVs and incubated with Alexa Fluor 546-labeled Nup120βM or Nup133α6. The CtNdc1 (242-646) fragment additionally harbored the sixth transmembrane helix of CtNdc1 to ensure proper reconstitution into GUV membranes. Source data are available for this figure: SourceData FS1.