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Fig. 1. Generation of knock-in animals expressing Erbb2 tyrosine phosphorylation
mutants. The knock-in targeting vector was constructed such that exon 1 of the
mouse Erbb2 gene was replaced with either (A) a rat Erbb2
cDNA or (B) a cDNA encoding a mutant Erbb2 receptor harboring the
Y1028F mutation. The cDNA is followed by a PGK-Neomycin/SV40-polyA
expression cassette and is targeted to the endogenous Erbb2 locus by
homologous 5' and 3' flanking arms, placing it directly under the
transcriptional control of the endogenous Erbb2 promoter. The
targeted allele introduces an additional HindIII site that was used
to distinguish between the endogenous wild-type allele (7.5 kb fragment) and
the targeted allele (4.0 kb fragment) in Southern blot analyses (A, inset).
(C) Schematic representation of the Erbb2 receptor depicted with the five
tyrosine autophosphorylation sites in the C-terminal tail and with the
corresponding amino acid number. Note that the numbering of the amino acids is
based on the rat Erbb2 sequence and will be referred to herein by these
designates. Also shown are the three individual tyrosine-to-phenylalanine
mutations described in this report: Y1028F, Y1144F and
Y1227F. (D) Alignment of the amino acid sequences surrounding
tyrosine 1028 in Erbb2 and tyrosine 992 in Egfr. (E) Expression analysis of
the knock-in allele. Total protein lysates prepared from E12.5 wild-type and
Erbb2 cDNA knock-in embryos were used to detect Erbb2 levels.
Detection of Grb2 protein (lower panel) served as an internal loading
control.
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