Figure 3.

Nesprin-1 mutants affect the interaction between nesprin-1α₂ and lamin A/C or SUN2. GST-tagged WT nesprin-1α₂ SR1-6 (equivalent to nesprin-1 giant SR 69–74) and mutants are generated and shown schematically (3A left panel, labelled with *), which constructs consisted of 837 amino acids, lacking KASH domain, equivalent to amino acids 7875–8662 of nespin-1 giant. GST pull-down using either GST-WT or mutant nesprin-1 beads showed all three mutants affected binding between nesprin-1 and lamin A/C or SUN2, but not emerin (A, B), which was confirmed by reverse GST pull down by transfecting either GFP-nesprin-1α₂WT or each mutant and using either GST-lamin A (amino acids 356–665) (C) or GST-emerin (amino acids 1–176) beads (D). The binding for each mutant was quantified by densitometry with respect to the input material and expressed as a ratio of the value obtained for WT protein. Three independent experiments were performed shown as mean ± SEM, *P < 0.05 using one-way ANOVA analysis. Coomassie blue staining gel also showed equal amount of GST-nesprin 1α₂, lamin or emerin beads used.