Plasmid-based Manipulation of SUN1 and Nesprin-2 in HeLa Cells

Plasmids encoding human full length SUN1 (GFP-SUN1) and the siRNA resistant (R) GFP-SUN1^R as well as GST-SUN1-NT (residues 1-239) have been described previously (SUN1 Accession number O94901)^{8 (link)}. Mutations to generate GFP-SUN1 or GFP-SUN1^R S110A, S113A, S110A/113A and S113D and GST-SUN1-NT S110D, S113D, S110D/S113D were introduced using a site-directed mutagenesis kit (Promega). GFP-SUN1^R-ΔC contains residues 1 to 412 encompassing the N-terminus and the transmembrane region but lacking the coiled coil region and the SUN domain. Transfections of HeLa cells were carried out using Lipofectamine 2000 (Invitrogen) or o-fekt (Bio-Budget Technologies, Krefeld, Germany). Analyses were done 24 to 48 hours after transfection. For siRNA-mediated depletion, HeLa cells were transfected with SUN1 targeted siRNA or control siRNA described previously^{8 (link)} and SUN2 targeted siRNA (GE Healthcare Dharmacon). For RNAi transfection, the reagent INTERFERin (Polyplus) was used. Cells were analyzed 72 to 96 hours after transfection. For rescue experiments, cells were transfected with GFP-SUN1^R or GFP-SUN1^R-ΔC plasmids using Lipofectamine 24 h after siRNA treatment. Analyses were done 72 to 96 hours after siRNA treatment^{8 (link)}.
sh RNA knockdown of Nesprin-2 was carried out using oligonucleotides targeting N- and C-terminal sequences of Nesprin-2 Giant as described^{19 (link), 20 (link)}.