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Fig. 11. Association between cyclin D3 and GSK-3ß. (A) In vivo. Reh cells were treated with or without forskolin (100 µM) and harvested at the indicated times. Whole cell extracts were prepared and immunoprecipitated (IP) with anti-cyclin D3 antibodies. The immunoprecipitates were then subjected to western blot analysis (W) with the indicated antibodies. The right-most lane shows the background where cell extracts obtained from control cells were immunoprecipitated with antibodies against cyclin A. The blot shown in the upper panel is the representative of four independent experiments. Lower panel, the results in each experiment were quantified using a densitometer and the densitometric values of GSK-3ß were normalized with those of cyclin D3. The values obtained were then plotted with the value for cells not treated with forskolin set as 100%. Vertical bars indicate the mean ±s.e. (B) In vitro. 2 µg of bacterially expressed His-cyclin D3 (wt) or His-cyclin D3 (T283A) proteins bound to Ni-NTA agarose beads or Ni-NTA agarose beads alone (upper panel) were incubated with whole cell extracts (600 µg) from Reh cells stimulated with forskolin (100 µM) for 2 hours or cells left unstimulated. The bound proteins were resolved on SDS-PAGE and analyzed by immunoblotting with anti-GSK-3ß antibodies (middle panel). The blot shown is the representative of four independent experiments. The results in each experiment were quantified and plotted as described in A (lower panel). (C) 2 µg His-cyclin D3 (wt) protein bound to Ni-NTA agarose beads or Ni-NTA agarose beads alone (upper panel) were incubated with 0.6 µg purified GSK-3ß protein for 2 hours. The beads were then subjected to western blot analysis with anti-GSK-3ß antibody (lower panel). One representative experiment of three is shown. (D) Reh cells transfected with His-cyclin D3 (T283A) expression vector were treated with 100 µM forskolin or left untreated. His-cyclin D3 (T283A) was precipitated by incubating the cell extracts with Ni-NTA agarose beads and then analyzed by western blot analysis (W) with the indicated antibodies. The right-most lane (control) shows the background where cell extracts obtained from mock-transfected Reh cells were precipitated with Ni-NTA agarose beads. The blot shown is the representative of four independent experiments. The results in each experiment were quantified and plotted as described in A (lower panel).
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