Fig. 9. MYC-induced Stat5 activation and precocious lactation are specific to MYC induction from D12.5-D15.5 of pregnancy. (A) Hematoxylin and Eosin staining of mammary histological sections from MTB and MTB/TOM mice induced with doxycycline from D6.5-D9.5 of pregnancy and sacrificed (sac) at D9.5 or D12.5 of pregnancy, as well as from mice induced from D12.5-D15.5 and sacrificed at D15.5 or D18.5 of pregnancy. Results are representative of experiments performed on three separate sets of animals. (B) Expression of the Myc transgene, targets of MYC transcriptional activation Cdk4, Fbl and Shmt1, and the lactation markers Csnd and Pip were determined by northern hybridization analysis. Total mammary RNA was prepared from MTB and MTB/TOM mice induced from D6.5-D9.5 or D12.5-D15.5 of pregnancy. (C) Western blot analysis of Stat5a/b Y694/Y699 phosphorylation. Mammary protein extracts were prepared from MTB and MTB/TOM mice induced from D6.5-D7.5, D6.5-D8.5 and D6.5-D9.5 or D12.5-D13.5, D12.5-D14.5 and D12.5-D15.5 of pregnancy. Proteins were separated by SDS-PAGE and blotted to nitrocellulose membranes. Blots were probed with antibodies specific to Y694/Y699 phosphorylated Stat5a/b, total Stat5a/b, and ß-tubulin. Results are representative of experiments performed on two separate sets of animals.