FIG. 7.
The half-life of Flag-tagged cyclin D1 protein is not changed by PC3. (A) Forty-five hours after cotransfection of NIH 3T3 cultures (2.3 × 105 cells in 60-mm-diameter dishes) with Flag-tagged cyclin D1 (2.15 μg) and either pSCT-β-Gal or pSCT-PC3 (2.15 μg each), cells were metabolically labeled for 2 h with [35S]methionine. Cells were washed with medium containing an excess of unlabeled methionine, collected, and lysed at the indicated times. Cell lysates containing equal amounts of proteins were then immunoprecipitated using the M2 monoclonal antibody against the Flag epitope. A control transfected with pSCT vector without Flag-tagged cyclin D1 is shown in lane 1. Shown are results of a representative experiment. (B) Graphic representation of Flag-tagged cyclin D1 expression. The data at individual time points are the amounts of 35S-labeled Flag-tagged cyclin D1 protein as measured by a PhosphorImager system and are the means ± SEM of four independent experiments. The half-lives of Flag-tagged cyclin D1 protein were calculated for each experiment by linear regression analysis of the density values at the different time points, transformed by common logarithm.