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Fig. 4. (A) Western blot of different human tissues using nesprin-2 N2, N3 and CH3 antibodies demonstrating tissue-specific and multiple isoforms in heart (H), kidney (K), skeletal muscle (S) and testis (T). Note the dominant small isoforms and the similar pattern of bands using the N2 antibody in heart and skeletal muscle and the multiple bands in testis; consistent with northern blot results. (B) Multiple isoforms were identified by all antibodies in vascular smooth muscle cells (V) and fibroblasts (F) with these cell types having identical isoform patterns on western blots. All bands were competed by excess peptide confirming antibody specificity. (C) 35S-labelled in vitro translation of nesprin-1
(asterisk), 2 (cross) and 2 ß(diamond) (first panel) and subsequent western blot analysis demonstrates specificity of N2 (middle panel) and N3 (end panel) antibodies. N2 recognised nesprin-2 and -2ß while N3 recognised only nesprin-2ß. Neither antibody recognised the closely related nesprin-1. Luciferase (
) (D) 35S-labelled in vitro translation of nesprin-1 and nesprin-2 CH domains (left panel) and western blot using the CH3 antibody (right panel) shows that CH3 recognises only the nesprin-2 domain and not the closely related nesprin-1 domain confirming antibody specificity. Multiple unlabelled bands on western blots in C and D represent rabbit nesprin-2 isoforms present in the rabbit reticulocyte lysate. (E) Western blot using nesprin-2 N2, N3 and CH3 antibodies and sub-cellular fractions of human VSMCs to identify nesprin-2 isoforms in cytoplasmic and nuclear domains. Predicted sizes of isoforms identified from cDNA analysis are marked with arrowheads and it is probable that the N2 antibody identifies nesprin-2 and ß isoforms. (F) Control blot shows -SM actin, SF-2 and emerin to confirm cytoplasmic, nuclear and NE fractionation. Cyto, cytoplasmic; NS, nuclear soluble; NE, nuclear envelope.
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