HeLa Flp-In T-REx cell line 30 (link) was stably transfected with an inducible plasmid expressing EmGFP and a microRNA against the ORF of the Nup358 mRNA using the BLOCK-iT Inducible Pol II miRNA RNAi Expression Vector Kit w/EmGFP from Life Technologies (with a modified Gateway destination vector compatible with the Flp-In system 4 (link)). Cells were treated for 4 days with 1 μg/ml of tetracycline to induce the expression of EmGFP and the miRNA. Isolation and plunge freezing of nuclear envelopes was carried out as previously described 4 (link). All cell lines used in this study have been regularly tested for Mycoplasma contamination but have not been authenticated.
Nup188, Nup205, Nup93, Nup155, Nup85 and Nup62 cDNAs were purchased from the human ORFeome collection, subcloned into a Gateway destination vector with an N-terminal His6-HA-StrepII-tag, and stably transfected into the cell line 293 Flp-In T-REx (Life Technologies). Cells were treated for 8 days with 1 μg/ml of tetracycline to induce the expression of the fusion protein, which was subsequently affinity-purified.
Nup188, Nup205, Nup93, Nup155, Nup85 and Nup62 cDNAs were purchased from the human ORFeome collection, subcloned into a Gateway destination vector with an N-terminal His6-HA-StrepII-tag, and stably transfected into the cell line 293 Flp-In T-REx (Life Technologies). Cells were treated for 8 days with 1 μg/ml of tetracycline to induce the expression of the fusion protein, which was subsequently affinity-purified.
