PDI and PDIp are involved in the remodeling of the conserved disulfide bridge in HShh. (A) HShh-HA and FLAG-tagged Trx-like ER proteins, in which one of their CXXC motifs was changed to CXXA, were coexpressed in 293T cells. Cell extracts were subjected to immunoprecipitation (IP) with HA or FLAG antibodies followed by SDS-PAGE and immunoblotting (IB) with FLAG and HA antibodies. Where indicated, the immunoprecipitated samples were reduced with DTT before electrophoresis. (B) Wild-type (WT) HShh-HA or the processing-defective C198A mutant was coexpressed with a FLAG-tagged CXXA mutant of PDI (C56A) in 293T cells. The cells were pulse labeled with [³⁵S]methionine for 3 min and chase incubated for different time periods. The proteasome inhibitor epoxomicin (1 µM) was added 1 h before the pulse. All samples were subjected to immunoprecipitation with HA antibodies followed by SDS-PAGE and fluorography. Where indicated, the samples were reduced with DTT before electrophoresis. Molecular masses are given in kilodaltons.