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Fig. 6. A, time course showing effect of CHX on cyclin D1 ({bullet}) and ß-actin ({blacksquare}) protein levels. CHX (final concentration, 100 µg/ml) was added at T=0 to block protein synthesis, and levels of cyclin D1 and ß-actin protein were quantified by Western blotting. Each point represents the mean of three different cultures ± SE. The mean for the zero time point was set at 1.0. Regression analysis indicated that the slope of the cyclin D1 decay curve differed significantly from zero (P < 0.0001), whereas the slope of the ß-actin decay curve did not (P=0.0932). The half-life of cyclin D1 protein was also calculated by regression analysis. B, effect of 15d-PGJ2 on cyclin D1 protein degradation as determined by the pulse-chase method. Cells were pulse-labeled with [35S]methionine for 1 h, washed three times, and transferred to chase medium containing 2 mM unlabeled methionine at T=0 (see "Materials and Methods"). Cyclin D1 protein was immunoprecipitated at the indicated chase times and quantified by SDS gel electrophoresis and autoradiography. The level of labeled cyclin D1 protein at the beginning of the chase (T=0) was set at 1.0. Each data point represents the mean of results obtained in two different experiments. Circles and solid line, control cells; triangles and broken line, cells treated with 15d-PGJ2. The half-life of cyclin D1 protein was calculated by regression analysis.