Fig. 10. Analysis of subcellular localization of cyclin D3 and GSK-3ß. (A) Reh cells were pretreated with the proteasome inhibitor MG-132 (10 µM) for 30 minutes before addition of forskolin (100 µM). Cells were cytospun onto coverslips at 2 hours after addition of forskolin, fixed in paraformaldehyde, and subjected to immunofluorescence microscopy after staining with cyclin D3 or GSK-3ß antibodies. The nuclear and cellular morphologies were visualized by Hoechst staining and phase contrast microscopy, respectively. One representative experiment of four is shown. (B) Reh cells were cultured in the presence or absence of 100 µM forskolin and harvested at the indicated time points. Subcellular fractions were prepared as described in Materials and Methods. Equal amounts of protein were resolved by SDS-PAGE and detected by immunoblotting with antibodies against GSK-3ß, actin and AKAP95. The blot shown is the representative of three independent experiments.