Overexpression of activated NeuNT in mammary tumors. (A) Using a probe that spans the transmembrane domain of Neu, an RNase protection reveals that MMTV-Cre Flneo NeuNT tumors (TM1–4) overexpress the activated form of neu. The mammary gland from a virgin MMTV-Cre Flneo NeuNT mouse (5801) was included, as was a positive control known to overexpress an activated form of neu (NDL 1–4). The full-length protected fragment is shown at the band labeled WT Neu. The mutant alleles are labeled NDL Neu and NeuNT, corresponding to their activating mutations. Thirty micrograms of RNA was used, except in the control NDL 1–4 tumor sample, where 20 μg was used. pgk was included as an internal control for equal loading of the samples. Adj. Gl., adjacent mammary gland. (B) Blotting for Neu reveals that only the tumors have elevated levels of Neu. Grb-2 was used to control for equal loading of the 120 μg of protein in each sample. MP, multiparious. (C) After immunoprecipitating for Neu, a blot was probed for phosphorylated tyrosine (P-Tyr), which demonstrated that catalytically active Neu was overexpressed in the tumors. Protein (1.3 mg) was used in this immunoprecipitation.