FIG. 3.

ERp57 and PDI function coordinately with ERp29 to unfold Py in vitro. (A) VP1 digestion pattern. Crude Py was incubated with DTT, EGTA, and either BSA or an ER lumenal extract, followed by trypsin addition where indicated. The samples were subjected to SDS-PAGE, followed by immunoblotting with a VP1 antibody. (B) Purified Py was pretreated with either calmodulin, ERp57, ERp72, or PDI in the presence of EGTA. The samples were then incubated with an ERp29-enriched ER lumenal extract, supplemented with trypsin, subjected to SDS-PAGE, and immunoblotted with a VP1 antibody. A 10% input for the amounts of ERp57, PDI, and the ERp29-enriched ER lumenal extract used is shown. (C) As for panel B, except NEM-treated ERp57 and PDI were used where indicated. (D) NIH 3T3 cells transfected with either a rat ERp29 or an N-terminally FLAG-tagged rat ERp29 construct were treated with the DSP cross-linker or left untreated. The resulting cell lysates were subjected to immunoprecipitation using an antibody directed against the FLAG epitope conjugated to agarose (IP:FLAG). The precipitates, as well as the cell lysates (input), were subjected to SDS-PAGE and immunoblotted with antibodies against ERp57 and ERp29. (E) As for panel D, except antibodies against ERp72 and PDI were used instead of antibodies against ERp57.