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Fig. 8. His-cyclin D3 (T283A) associates with CDK4 and CDK6. (A) Reh cells were transfected with expression vectors encoding His-cyclin D3 (wt) or His-cyclin D3 (T283A). Following transfection, whole cell extracts were prepared and immunoprecipitated (IP) with antibodies against CDK4, CDK6 or with non-immune rabbit serum (NRS). The recovered proteins were resolved on SDS-PAGE and then subjected to immunoblotting with anti-CDK4, CDK6, or cyclin D3 antibodies. As positive control for CDK4, CDK6, and cyclin D3, 50 µg total cell lysates prepared from Reh cells and cells expressing His-cyclin D3 (T283A) were analysed in parallel (total lysate). (B) Reh cells were mock-transfected or transfected with His-cyclin D3 (wt) or His-cyclin D3 (T283A) vectors. Following transfection, whole cell extracts were prepared and incubated with Ni-NTA agarose beads to isolate His-cyclin D3 and its associated proteins. The recovered proteins were then separated by SDS-PAGE and immunoblotted with anti-CDK4, CDK6, or cyclin D3 antibodies. As positive control for CDK4, CDK6, and cyclin D3, 50 µg total cell lysates were prepared from mock-transfected Reh cells, and cells expressing His-cyclin D3 (T283A) were analysed in parallel (total lysates).
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