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. 2014 Nov 18;290(2):987–995. doi: 10.1074/jbc.M114.599852

FIGURE 1.

FIGURE 1.

Generation and characterization of hAlcα CTF transgenic mouse lines. A, construct for generation of hAlcα CTF transgenic mouse lines. The cDNA sequence encoding amino acids 817–971 of human Alcα1 (971 amino acids) was ligated with cDNA encoding the signal sequence (amino acids 1 to 28) and cloned into a vector with the 5′-PDGF-β promoter sequence and 3′-SV40 poly(A) sequence. DNA fragments prepared by digestion with SalI and NotI were used for injection into fertilized eggs. B, identification of human p3-Alcα secreted from HEK293 cells expressing hAlcα CTF. Amino acid sequence of human p3-Alcα35 and a representative MS spectrum of immunoprecipitate recovered from medium of HEK293 cells expressing human Alcα CTF with anti-p3-Alcα antibody UT135 are shown. Numbers in parentheses indicate p3-Alcα species (35 indicates p3-Alcα35, a major p3-Alcα species). Intens, Intensity; a.u., arbitrary unit. C, establishment of hAlcα CTF Tg mouse founders. A summary of the individual numbers at the respective experimental stages is shown. D, genomic Southern blot analysis of six transgenic lines. Genomic DNA of six founders was analyzed by Southern blotting with DNA fragments prepared by SalI/NotI digestion (A) as a probe. The transgenic lines used are numbered, along with non-transgenic mouse (Non-Tg) DNA and 10 copies of injected DNA. E, expression levels of hAlcα CTF in three Tg mice lines. Protein expression of hAlcα CTF, a transgenic product, was quantified by immunoblotting. A brain region, including the cerebral cortex and hippocampus of Tg18, Tg47, and Tg54 mice along with their non-Tg littermates (N), was lysed and analyzed by immunoblotting with anti-Alcα no. 958 (top) and anti-flotillin-1 (bottom) antibodies. Band densities of Alcα CTF were quantified and standardized with respect to the band density of flotillin-1. Expression levels in Tg mice (filled bars) are shown as a ratio relative to the endogenous Alcα CTF level of non-Tg littermates (open bars), which was set at 1.0. Data represent mean ± S.E. (n = 3). Specificity of the anti-Alcα no. 958 antibody is shown in Fig. 2A.