Fig. 1. The p97 adaptor, Ufd1–Npl4, mediates binding to polyubiquitylated proteins. Recombinant mammalian p97 (lanes 2–4), p47 (lane 5) and Ufd1–Npl4 (UN, lane 6) were biotinylated and immobilized on streptavidin–beads. The p97–beads were either used without further treatment (lane 2) or first saturated with recombinant p47 or UN (lanes 3 and 4, respectively). The immobilized protein complexes were then incubated with a Triton X-100 extract from HeLa cells and bound proteins analyzed by western blotting using a ubiquitin-specific antibody (upper panel). The filter was also stained with Ponceau S to visualize the immobilized proteins (lower panel). Note that only UN efficiently binds polyubiquitylated proteins (pUb) either alone or in combination with p97.