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. 2006 Aug;26(15):5688–5697. doi: 10.1128/MCB.00779-06

FIG. 2.

FIG. 2.

Generation of Grp78 KO mice. (A) Schematic drawings for the Grp78 cDNA, the WT allele, the tri-loxP-targeting vector, the targeted tri-loxP (T), and the KO alleles. The exons encoding the ATPase domain and peptide-binding domain of GRP78, the location of the insertion of the floxed neo cassette, the three loxP sites (▸), and the pgk-TK expression cassette (TK) are indicated. The location of the primers [→] used in the PCR genotyping, the external 5′ probe A and the external 3′ probe B used in the Southern blot, and the BamHI restriction sites [B] are also indicated. (B) Southern blot of BamHI-digested wild-type (+/+), heterozygous Grp78+/T, and heterozygous Grp78+/− DNA using the 5′ probe A. The size of each band (in kilobases) is indicated. (C) Southern blot of BamHI-digested DNA from wild-type (+/+) and Grp78+/− siblings, using the 3′ probe B against the 3′-adjacent Rab9p40 (Rab) gene (GenBank accession no. NM_145522). (D) Whole-cell lysates from livers of wild-type (+/+) adult mice and heterozygous Grp78+/− siblings were subjected to Western blot analysis with anti-GRP78 (N20) antibody. The film was overexposed to detect any truncated form of GRP78, if it exists. The asterisk indicates the 78-kDa full-length GRP78 protein. (E) Total RNA from WT (+/+) or heterozygous (+/−) E10.5 embryos were subjected to Northern blot analysis to analyze the Rab transcript level. The 28S rRNA was used as an RNA loading control.