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Fig. 12. Regulation of GSK-3ß activity by forskolin. (A) Reh cells were treated with or without forskolin (100 µM) and harvested at the indicated times. Whole cell extracts were prepared, immunoprecipitated with GSK-3ß antibodies and assayed for activity towards Tau. The specificity of the kinase reactions was examined by assaying the GSK-3ß activity recovered from control cells in the presence of 50 µM LiCl, or by immunoprecipitation of control cell extracts with non-immune mouse serum (NMS) (upper panel). The activities were then expressed as a percentage of the activity of GSK-3ß recovered from untreated cells (100%) and are mean ±s.e. of five independent experiments (lower panel). (B) Reh cells were treated as in A. Whole cell extracts were prepared, immunoprecipitated with GSK-3ß antibodies and assayed for activity towards GST-cyclin D1. The specificity of the kinase reactions was examined as described in A (upper panel). The activities were then plotted with the activity of GSK-3ß recovered from control cell set as 100% (lower panel). (C) Reh cells were treated as in A, total cell lysates were prepared and then subjected to immunoblotting with antibodies against GSK-3ß, GSK-3ß (Ser-9) and actin. The immunoblotblot shown is the representative of four independent experiments.
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