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Fig. 6. Gie associates with tubulin independent of its guanine-nucleotide-bound forms or the absence of its effector domains. (A) Extracts from HeLa cells expressing FLAG-tagged wild-type Gie1 and Ha-Ras were subjected to immunoprecipitation (IP) with the anti-FLAG antibody. The precipitants were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (left) or immunoblotting (IB) with an anti-ß-tubulin antibody (right). The asterisk denotes the position of interacting protein. (B) Microtubule co-sedimentation assay. Gie stayed mainly soluble (sup) when microtubule polymerization was inhibited by keeping on ice (lanes 2,4), and was specifically recovered in the pellet (pell) in the presence of Taxol-stabilized microtubules (lanes 1,3). The behavior of the Rab5 is shown as a negative control. (C,D) HeLa cells were transfected with expression vectors encoding the proteins listed at the bottom and the cell extracts were immunoprecipitated with the anti-FLAG antibody. The precipitants were subjected to immunoblotting using anti-FLAG and anti-ß-tubulin antibodies.
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