Figure 4.

BiP releases ERdj3 from D-Luc in an ATP-dependent manner. (A) Chemically denatured luciferase was added to the wells followed by ERdj3 binding as described earlier. After washing, the indicated amounts of WT BiP were added to the ERdj3–luciferase complexes with (hatched bar) or without (solid bar) ATP and incubated for an additional 1 h at room temperature. After washing, the amount of ERdj3 that remained bound to luciferase was detected with an anti-ERdj3 antiserum. (B) On a parallel plate, the amount of BiP that was associated with luciferase was determined by incubating with an anti-BiP antiserum. (C) Either wild-type or mutant (R197H) BiP was allowed to bind to luciferase. After washing, ERdj3 was added to half the wells and the amount of BiP that was bound without ERdj3 (solid bars) or with ERdj3 (hatched bars) was measured with an anti-BiP antiserum. In a parallel set of wells, ERdj3 binding was measured with an anti-ERdj3 antibody (checkered bars).