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Fig. 2. Forskolin increases the rate of cyclin D3 turnover. (A) Reh cells were pretreated with cycloheximide (25 µg/ml) for 15 minutes followed by forskolin (100 µM) over an 80 minute 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 and actin antibodies. The blot shown is the representative of five independent experiments. (B) The immunoblot in A was scanned and the intensity of the protein bands was quantitated and plotted on a semi-log graph with the value obtained for cells not treated with cycloheximide set as 100%. The values were normalized with those of actin. (C) Freshly isolated human B cells were stimulated with a combination of anti-µ and SAC for 32 hours as described in Materials and Methods. Cells were then treated as in A for the indicated times and analysed for the expression of cyclin D3 and actin by western blotting.
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