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Fig. 1. Regulation of cyclin D3 expression and phosphorylation by forskolin. (A) Reh cells were cultured in the presence or absence of forskolin (100 µM) and harvested at the indicated times. Total RNA was recovered, and 15 µg of the RNA from each sample was analyzed by northern blotting using ³²P-labelled cyclin D3 probe as described in Materials and Methods. The ethidium bromide-stained 18S RNA was used as a loading control (lower panel). (B) Reh cells were treated as in A, lysates were prepared and then subjected to immunoblotting with antibodies against cyclin D3 and actin. The immunoblot shown is the representative of five independent experiments. (C) Reh cells were treated with or without forskolin (100 µM) for 1 hour. Cells were lysed in buffer A, and the cell lysates were then treated with CIP in the presence or absence of PPI for 15 minutes at 30°C as described in Materials and Methods. The lysates were then resolved on SDS-PAGE, and cyclin D3 was detected by immunoblotting. The immunoblot shown is the representative of four independent experiments.

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