Figure 3. Loss of Rheb1 inhibits ErbB2-mediated 4EBP1 phosphorylation in Mammary Epithelium.

(A) Representative images of immunofluorescence and immunohistochemistry staining of mammary ductal structures from Rheb1^wt/wt NIC and Rheb1^fl/fl NIC mice at tumor endpoint for indicated antibodies. Scale bars represent 20 μm. (B) Representative images of immunohistofluorescence and immunohistochemistry staining of mammary ductal structures from Rheb1^wt/wt MIC and Rheb1^fl/fl MIC mice after 14 days of Dox induction for indicated antibodies. Scale bars represent 20 μm. (C) Grading scheme of phospho-4EBP1 S65 IHC staining intensity in Cre-positive mammary epithelium. Scale bar represents 50 μm. (D) Graph depicting frequency of grade 1, 2 and 3 p4EBP1 S65 staining in Cre-positive mammary epithelium in adjacent mammary glands of Rheb1^wt/wt NIC and Rheb1^fl/fl NIC mice at tumor endpoint (top), and Rheb1^wt/wt MIC and Rheb1^fl/fl MIC mice after 14 days of Doxycycline induction (bottom). Values represent mean (+/− SEM) frequency of each grade within minimally 150 Cre-positive cells from at least 3 biological replicates from each genotype. Statistical significance was assessed by two-tailed unpaired Student’s t-test.