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Fig. 9. His-cyclin D3 (T283A) expression abrogates the inhibitory effect of forskolin on CDK6 kinase activity. Mock-transfected Reh cells or cells that were transfected with His-cyclin D3 (wt) or His-cyclin D3 (T283A) vectors were treated with or without forskolin (100 µM). Two hours after treatment cells were harvested and lysed. One aliquot of each whole cell extract (600µg) was immunoprecipitated (IP) with 2 µg anti-CDK6 antibody or non-immune rabbit serum (NRS) and utilized for an in vitro CDK6 kinase assay (upper panel). A second aliquot of each whole cell extract was immunoprecipitated with anti-CDK6 antibody or NRS. The recovered proteins were separated on SDS-PAGE and then subjected to western blotting (W) with CDK6 (middle panel) or cyclin D3 (lower panel) antibodies. The results shown are representative of three independent experiments.





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