Bradford colorimetric protein assay

Whole-cell protein extracts were obtained from the mammary glands of virgin, 17.5-day pregnant female mice or from mammary tumors using standard techniques. The protein content was determined by the Bradford colorimetric protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Total protein extracts (40 μg) were subjected to SDS/PAGE. The separated proteins were transferred to Amersham nitrocellulose membranes and probed with the antibodies listed in Table S1.
The blots were visualized with the Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) using a ChemiDoc MP System and the ImageLab software (Bio-Rad, Hercules, CA, USA).

In those tumors that were large enough, we froze part of the tumor in liquid nitrogen at the moment of the sacrifice for western blot analysis. Whole-cell protein extracts were subjected to SDS/PAGE using standard techniques. Protein content was determined by the Bradford colorimetric protein assay (BioRad Laboratories; Hercules, CA; USA). The antibodies used in western blots were against p53 (NCL-p53-CM5p, Novocastra, Leica Biosystems, NewCastle, UK); Stat3 (phospho tyr705; 9131), Stat3 (4904); Akt (phospho ser473; 4058) (Cell Signaling Technology, Danvers, MA, USA); p19^ARF (ab80) (Abcam, Cambridge, UK); Akt1/2 (sc-1619), p16^ink4a (sc-1207), Cyclin D1(sc-753), and actin (sc-1616) as a loading control (Santa Cruz Biotechnology, Santa Cruz, CA, USA).