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Fig. 2. Optimal interaction of TOM1 with clathrin requires residues 300-321 and 362-366. (A) Schematic diagram of TOM1 deletion constructs used in the clathrin pull-down experiments. VHS, VPS-27, Hrs and STAM; GAT, GGAs and TOM1 homology. (B) Residues 300-366 are required to interact with clathrin. Glutathione-sepharose-bound recombinant GST fusion proteins of the deletion constructs depicted in A were incubated with A431 cytosol and proteins bound to the beads and input were resolved by SDS-PAGE analysis followed by western blotting with anti-clathrin heavy chain antibodies. GST and GST fusions from the same gel were visualized by Coomassie Blue staining as a control for levels of fusions used in the pull-down experiments. 50 µg input cytosol and proteins from post-pull-down extracts were precipitated with TCA and similarly analyzed by SDS-PAGE and immunoblotting with anti-clathrin heavy chain and anti-ß-tubulin antibodies, the latter to indicate loading levels.
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