Fig. 2.

Generation, genotyping, and characterization of Car5B KO mice. (A) Structure of the Car5B gene (Top), the targeting construct (Middle), and the predicted structure of the disrupted Car5A gene after homologous recombination and excision of the neomycin-resistance (Neo) cassette (Bottom). The open box indicates the position of the external probe. Only the relevant restriction sites are shown. The numbered solid boxes represent exons. Neo and TK (thymidine kinase) refer to positive and negative selection markers, respectively. (B) Genotyping of the Car5B KO colony. Genotyping by PCR was done on mouse-tail genomic DNA. PCR products present in WT (0.52 kb) and mutant (0.44 kb) mice were present in the heterozygotes. (C) Car5B transcripts in liver and kidney amplified from WT mice but not from KO mice. (D) Western blot analysis. The membrane proteins (50 μg) from liver and kidney of CA VB WT and KO mice were electrophoresed on a 12% (wt/vol) SDS-PAGE gel. After blotting, the membranes were probed with rabbit anti-mouse CA VB antibodies. A 31-kDa band present in both liver and kidney from the WT animals was absent in the same tissues from KO animals.