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The dominant lethal BiP mutants are unable to bind to the Sec63p DnaJ domain. GST–63Jp was prebound to glutathione–cross-linked agarose, and BiP and either ATP or ADP were added. Proteins remaining in the supernatant (S) or associated with the pellet (P) were resolved by SDS-PAGE and visualized by Coomassie brilliant blue staining. Signals corresponding to BiP and GST–63Jp are indicated, as are molecular weight markers (M; × 10−3).
