Fig. 5.

TorsinA associates with the KASH domain. (A) Human 293T cells were transfected with lentivirus vector encoding expression constructs for GFP-mouse-nesprin-3α or GFP-mouse-nesprin-3αΔ KASH. After 48 hours the cells were lysed and proteins immunoprecipitated with antibodies to torsinA or IgG (no added ATP), resolved by SDS-PAGE and immunoblotted with antibodies to plectin, GFP, vimentin, torsinA and GAPDH. Similar amounts of torsinA were precipitated from non-transfected cells and cells transfected with the GFP-nesprin-3α or GFP-nesprin-3αΔ KASH constructs. By contrast, no detectable plectin was immunoprecipitated with torsinA from cells expressing GFP-nesprin-3αΔ KASH, although some was immunoprecipitated from cells expressing GFP-nesprin-3α. (B) A similar experiment to A except that cells were transfected with expression constructs for GFP-KASH-3α, GFP-KASH-1 or GFP-KASH-2 (i.e. the KASH domains of nesprin-3α, nesprin-1 and nesprin-2, respectively). Immunoprecipitation with torsinA antibodies followed by immunoblotting for GFP revealed an association of torsinA with the KASH domains of all three nesprins. The percentage of each protein immunoprecipitated from lysates by torsinA antibodies is: 11±3% for GFP-KASH-3α, 21±5% for GFP-KASH-1, 12±4% for GFP-KASH-2 (values are mean percentage ± s.e.m., n=3 experiments).