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Fig. 1. Generation of Nesprin-2-Giant KO mice. (A) Diagram illustrating the partial genomic organization of the Nesprin-2 (WT) locus coding for the ABD domain (yellow boxes), the targeting construct and the targeted allele (mutant allele). The Nesprin-2-Giant ATG translation start site in exon 1 is indicated with an arrow. The location of the 5' and 3' probes used for Southern blot analysis is also indicated. Primer sets correspond to those used in PCR analysis for identifying the WT (1.2 kb, Primer set 1: 5'-CATAGAACATGCCCTGACATTCCTG-3' and 5'-CTGTTTCTGAGTATTGCATGCGCTTG-3') and mutant (1.1 kb, Primer set 2: 5'-TGTCTGCCTACATGTTACTATGGTC-3' and 5'-TGCGAGGCCAGAGGCCACTTGTGTAGC-3') alleles. Primers used for RT-PCR are indicated (see Fig. 9 for more details). Ex, exon; E, EcoRI; S, SacI; K, KpnI. (B) Southern blot analysis of mouse-tail DNAs digested with EcoRI for 5' analysis and SacI for 3' analysis. (C) PCR analysis of DNAs isolated from Nesprin-2^+/+, Nesprin-2^+/– and Nesprin-2^–/– mice. (D) Western blot analysis of HaCaT (human keratinocytes), and primary Nesprin-2^+/+ and Nesprin-2^–/– (clone 1 and 2) fibroblast homogenates. Blotting with pAbK1 indicates the absence of Nesprin-2 Giant in KO fibroblasts (arrowhead) and the presence of Nesprin-2 C-terminal isoforms (asterisks). For HaCaT cells, ECL signals were obtained after 5-minutes exposure; for dermal fibroblasts, signals were obtained after prolonged detection. (E) Structural features of all known Nesprin-2 isoforms. Major domains are indicated in different colours and shapes. The epitopes and identity of various anti-Nesprin-2 antibodies used are indicated (inverted Y).

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