Figure 5.

p58^IPK interacts with a secretory protein in the ER lumen. (A) Preprolactin was synthesized in vitro by using rabbit reticulocyte lysate and pancreatic microsomes. Untranslocated (pPrl) and translocated, signal sequence-cleaved material (Prl) are indicated. The microsomes were isolated by centrifugation, solubilized under nondenaturating conditions, and subjected to immunoprecipitation by using antibodies against PDI, p58^IPK, BiP, the β subunit of α glucosidase II (also known as 80K-H), and the HA epitope tag. Aliquots of the total translation products (lane 1), the isolated microsomes (lane 2), solubilized microsomes (lane 3), and immunoprecipitates were analyzed by SDS-PAGE and autoradiography. (B) Preprolactin was synthesized in vitro by using rabbit reticulocyte lysate and pancreatic microsomes as described above and adjusted to either 1% Triton X-100 (native conditions; lanes 1–5) or 1% SDS (denaturing conditions; lanes 6–10). After incubation of the denaturing sample at 37°C for 10 min, it was adjusted to 1% Triton X-100 and 0.1% SDS. Both samples were subjected to immunoprecipitation with the indicated antibodies. Aliquots of the input (lanes 1 and 6) and immunoprecipitates are shown.