Fig. 1

Generation of Caskin1-KO mice (a) Knockout strategy for the Caskin1 gene. Homologous recombination of the targeting plasmid resulted in insertion of the pgk-neo cassette (neo) and loxP sequences (filled triangles) into introns 1 and 6 of Caskin1. Floxed mice obtained following germline transmission of ES cells harboring the homologous recombination (targeted) were crossed with “Cre-deleter” mice. Exons 2–6 of the caskin1 gene, together with the neo cassette, were deleted from floxed mouse, causing a translational frameshift. A: AflII, EV: EcoRV, H: HindIII, K: KpnI, X: XbaI. (b) Southern blot analysis of homologous recombination in ES cells. Genomic DNA was prepared from wild-type (+/+) and Caskin1 lox/+ ES cells. (Left) HindIII-digested DNA hybridized with a 5′ probe yielded a 12.7-kb band for the wild-type and a 9.1-kb band for the floxed allele. (Middle) AflII- or KpnI-digested DNA hybridized with a 3’arm probe yielded, respectively, a 16.5- or 23.5-kb band for the wild type and an 18.3- or 19.8-kb band for the floxed allele. (Right) EcoRV-digested DNA hybridized with a Neo probe yielded a 11.5-kb band for the for floxed allele. (c) C-terminal amino acid sequences of mouse Caskin1 and Caskin2. The red and blue sequence indicate the antigen sequences used for generation of the anti-Caskin1 and anti-Caskin1/2C antibodies, respectively. Symbols: “*”, universally conserved residue that this position; “:”, strong similarity is fully conserved; “.” weak similarity is fully conserved. d Western blot analysis of Caskin1 expression in spinal dorsal horn (DH) and hippocampus (Hippo) in wild-type (WT) and Caskin1-KO (KO) mouse tissues. Total proteins (20 μg/lane) were resolved on a 7.5% SDS-PAGE gel, and Caskin1 was detected with anti-Caskin1/2C antibody after blotting to a PVDF membrane