Fig. 2. The hEGFR^KI allele is not efficiently expressed in epithelial tissues. (A) Expression of the endogenous mouse Egfr and the hEGFR^KI mRNAs measured by RNAse protection assay. The analysis was performed on total RNA isolated from various organs of a heterozygote hEGFR^KI/+ male mouse employing an antisense Egfr riboprobe, which detects both the endogenous mouse Egfr and the hEGFR^KI transcripts. In addition to 93 bp of nonspecific sequence (broken line), this riboprobe encompasses the region of the hEGFR^KI allele bridging the Egfr mouse promoter, the loxP site and the 5' end of the hEGFR cDNA. The input probe and the protected fragments are depicted schematically and the black triangle indicates the loxP site. A mouse S16 riboprobe (lower panel) was used as an internal control for equal sample loading in each lane. (B) Western blot analysis showing EGFR protein expression in brain and liver of mice of different genotypes. Immunoblotting was performed on total protein extracts using an anti-EGFR antibody recognising human and mouse EGFR and anti-tubulin was used as a control for protein loading. (C) In vitro EGFR autophosphorylation assay measuring EGFR protein levels. Prior to the kinase assay, protein lysates from various organs were immunoprecipitated with an anti-EGFR antibody recognising both the mouse and the human EGFR protein. Equal loading of protein was verified by Coomassie Blue staining (data not shown)