Research Article

Cytoskeletal Interactions at the Nuclear Envelope Mediated by Nesprins

Figure 3

Nesprin-1 N-terminal sequences interact with Nesprin-3. (a) COS7 cell lysates expressing the N-terminal Nesprin-3 (GFP-Nesprin-3-ΔC) fusion protein were incubated with the indicated Nesprin-1-165 polypeptides fused to GST. GST was used as control. The cell lysates (input and supernatant, upper and middle panel) and the pelleted protein (lower panel) were subjected to SDS-PAGE followed by western blotting using mAb K3-184. P, pellet; S, supernatant. (b) Yeast-two hybrid analysis of the interaction between Nesprin-1 and Nesprin-3. Fragments corresponding to Nesprin-1-165 full length and the indicated Nesprin-1-165 polypeptides were fused to the Gal4 DNA binding domain (BD), the N-terminal part of Nesprin-3 lacking the KASH domain (Nesprin-3-ΔC) was fused to the GAL4 activation domain (AD). The corresponding plasmid pairs were cotransformed into yeast strain Y190 and interaction was assessed by filter lift β-galactosidase assay. Blue colour development indicates β-galactosidase activity.
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