Figure 5.

Binding of clathrin heavy chain to GST–CALM. GST–CALM, GST–1–413, and GST–414–652 were bound to glutathione-agarose beads and incubated with the cytosolic (saponin extract; A) or membrane (TX100 and deoxycholate extract; B) fractions of ³⁵S-metabolically labeled HeLa cells. Beads alone or with immobilized GST were used to control for the nonspecific binding of proteins. The agarose precipitates were resolved via 7.5% SDS-PAGE and transferred to a nitrocellulose membrane. Top, autoradiographs of the membranes are presented. The position of a 175-kDa–labeled band specifically associated with GST–CALM and GST–414–652 is indicated by arrows. Bottom, the same membranes were probed by Western blotting with antibody TD.1 to CHC. The detected band precisely overlapped with the 180-kDa radioactive band. The blots are from the same experiment. Asterisks mark other unidentified bands specifically bound to the fusion proteins.