Fig. 7. STAT5 activation is abolished in Erbb4^Flox/FloxWap-Cre mammary glands. Immunohistochemical analysis of biparous control Erbb4^+/+Wap-Cre (A,C,E,G) and Erbb4^Flox/FloxWap-Cre (B,D,F,H) paraffin wax-embedded mammary glands, at P13.5 (A,B), P17.5 (C,D) and L1 (E,F), for STAT5 activation using an affinity-purified antibody directed against STAT5 phosphorylated at the regulatory amino acid Y694 (A-F; P-STAT5). A STAT5 antibody was used in immunohistochemistry of paraffin wax-embedded mammary glands to identify both phosphorylated and inactive STAT5 populations (G,H; STAT5). The inset in B is a higher magnification view of positive P-STAT staining. Arrowheads and arrows indicate positive nuclear and cytoplasmic staining, respectively. Scale bar: 50 µm. (I) Expression of inactive STAT5 in Erbb4^Flox/FloxWap-Cre mammary glands was confirmed by western blot analysis of mammary gland protein lysates. STAT5 and ERBB4 were immunoprecipitated from Erbb4^+/+Wap-Cre control and Erbb4^Flox/FloxWap-Cre mammary gland protein lysates prepared from biparous mice at L1. ERBB4 immune complexes were probed with an ERBB4 antibody (I; ERBB4/ERBB4). Phosphorylation of immunoprecipitated STAT5 protein was determined by western blot analysis using a phosphotyrosine antibody (I; STAT5/P-tyr) and an antibody specific for STAT5 phosphorylated at Y694 (I; STAT5/P-STAT5). The relative epithelial cell population was determined by probing 50 µg of total mammary gland lysate with an antibody specific for keratin 18 (I; NA/K18).