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Fig. 4. Binding of MBP-torsinA and MBP-torsinAE recombinant proteins to nesprin-3 and GST KASH. (A) MBP, MBP-torsinA or MBP-torsinAE fusion proteins (Hewett et al., 2003) (8 µg) were bound to amylase resin (New England Biolabs) and incubated overnight at 4°C with lysates of wild-type MFs (500 µl protein in 1 ml) in RIPA buffer containing 1 mM ATP and 3 mM MgCl₂. Beads were washed five times with RIPA buffer and proteins bound to beads resolved by SDS-PAGE and immunoblotted with antibodies to nesprin-3 (top) and MBP (bottom), with relative binding assessed as in Fig. 1. MBP-torsinAE consistently pulled down threefold more nesprin-3 than did MBP-torsinA (3.0±0.3, n=3, P<0.003, ANOVA). The lower, immunoreactive nesprin-3 band is a degradation product, also seen by Wilhelmsen et al. (Wilhelmsen et al., 2005). (B) GST or GST-KASH fusion protein (10 µg) bound to glutathione-Sepharose beads was incubated with 10 µg of MBP-torsinA or MBP-torsinAE in 500 µl PBS containing 1 mM ATP and 3 mM MgCl₂ for 1 hour at 4°C. Unbound material was removed by centrifugation and the beads washed five times with 0.1% NP40 buffer followed by an additional wash with PBS. The proteins bound to the beads were resolved by SDS-PAGE and visualized by western blotting with antibodies to MBP and GST.

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