Fig. 4. Protein levels of cell cycle regulatory molecules in mammary tissues at different hormone conditions. A, total protein levels of cdk4, cdk2, cyclin D1, cyclin D2, p21Cip1, p27Kip1, and actin as control for equal sample loading. +Pit, pituitary isograft stimulated. B, histograms for the same proteins in A after normalization, the results of the protein content in 105 mammary epithelial cells of each group, and the results at each hormonal status. All proteins were analyzed by Western blot in 500 µg of tissue protein extract/sample. Antibodies were tested for specificity prior to use. The virgin/ovariectomized (OVX) sample is missing in the cdk4 panel. All protein bands were visualized by ECL detection reagent system after different exposure times against BioMax Kodak films. The intensity of the protein bands was scanned densitometrically using the Phosphorimager SF analyzer. Numbers on top of columns in B represent the number of fold increase or decrease compared with the internal control of each group. +pit, pituitary isograft stimulated.