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Fig. 7. Regulation of cyclin D3 stability depends on the integrity of Thr-283. (A) Reh cells transfected with His-cyclin D3 (wt) or His-cyclin D3 (T283A) vectors were cultured in the presence or absence of forskolin (100 µM) and harvested at the indicated time points. Total cell lysates were prepared and then subjected to immunoblotting with antibodies against cyclin D3. The immunoblot shown is the representative of three independent experiments. (B) Reh cells engineered to express His-cyclin D3 (wt) or His-cyclin D3 (T283A) were pretreated with cycloheximide (25 µg/ml) for 15 minutes followed by forskolin (100 µM) over a 2-hour time course. Cells were harvested at the indicated time points after addition of forskolin, lysates were prepared and equal amounts of protein were analyzed by immunoblotting with cyclin D3 antibodies. The blot shown is the representative of four independent experiments. Lower panel, the immunoblots were exposed, scanned and the intensity of the protein bands was quantified and plotted on a semi-log graph with the value obtained for cells not treated with cycloheximide set as 100%. (
) Endogenous cyclin D3, cycloheximide; (
) endogenous cyclin D3, cycloheximide + forskolin; (µ) His-cyclin D3 (wt), cycloheximide; (
) His-cyclin D3 (wt), cycloheximide + forskolin; (
) His-cyclin D3 (T283A), cycloheximide; (
) His-cyclin D3 (T283A), cycloheximide + forskolin.
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