FIG. 2.

GRP78/BiP binding is ATP dependent. Biotinylated HepG2 lysates were incubated with GST and further incubated with glutathione-Sepharose beads. The unbound proteins (precleared lysate; lane 1) were incubated with GST-pre-S1 fusion protein that bound to glutathione-Sepharose beads. After the beads were extensively washed with lysis buffer, the bound proteins were eluted with PBS (lane 2), ATP elution buffer (lane 3), and ATP elution buffer again (lane 4), in that order, before they were eluted by boiling in the protein sample buffer (lane 5). All eluates were divided into two parts and subjected to SDS-PAGE and Western blotting using goat anti-BiP and HRP-conjugated anti-goat IgG antibodies. Molecular size markers (in kilodaltons) (lane M) are shown on the left.