Figure 1.

Interaction of CVAK104 with clathrin. (A and B) Purified recombinant CVAK104 was incubated with the indicated amounts of clathrin cages with (A) or without light chains (B). Samples were ultracentrifuged and the resulting supernatants (S) and pellets (P) were analyzed by SDS-PAGE and Western blotting with a CVAK104 antibody. Note that an occasionally visible degradation product of CVAK104 fails to bind to the clathrin cages (asterisk in A). (C) Pull down of recombinant CVAK104 by recombinant clathrin TD fused to GST and immobilized to glutathione-Sepharose. Immobilized GST served as a control. S and P fractions were analyzed by SDS-PAGE and Western blotting. The results demonstrate that CVAK104 interacts directly with the TD of clathrin. In the cage binding assays (A and B), the loadings are directly comparable. In C, the pellet fractions were twofold concentrated compared with the supernatants.