Fig. 4. Comparison of Erbb2 protein and transcript levels in knock-in embryos. For immunoblot analyses, total protein was isolated from the different E12.5 embryos as indicated and subjected to SDS-PAGE. Where possible, control littermates were used for comparison analyses. Membranes were subsequently incubated with an anti-Erbb2 antibody (A,B, upper panel). Detection of Grb2 protein (A,B, lower panel) was used as a sample loading control. (C) Erbb2 protein levels were quantified by using ¹²⁵I-conjugated secondary antibodies and analyzed using PhosphoImager and ImageQuant software (Amersham Biosciences). The absolute levels of Erbb2 detected were normalized to Grb2 levels. The graph depicts relative levels of Erbb2 expressed as a percentage of Erbb2 levels in wild type embryos. (D) Erbb2 transcript levels (upper panel) were detected by RNase protection assays on total RNA isolated from E12.5 embryos. The mouse phosphoglycerate kinase (pgk) riboprobe (lower panel) was used as an internal control for equal sample loading. (E) The protein levels of all four ErbB family members (Egfr, Erbb2, ErbB3, and ErbB4) were also detected as described above for E12.5 embryos.