Figure 7.

The synthesis of BiP is highly induced by tunicamycin treatment in HeLa cells. (A) HeLa-RI₃₃₂ and HeLa-RI₃₃₂-Thr cells were left untreated (lanes a and e) or preincubated with tunicamycin (Tu; 5 μg/ml) for 1 h (lanes b and f), 2 h (lanes c and g), or 4 h (lanes d and h). Cells were pulse labeled in the continued absence or presence of the drug for 30 min. Cell lysis and immunoprecipitations with the monoclonal mouse anti-BiP antibody were performed as described in MATERIALS AND METHODS. Samples were analyzed by SDS-PAGE and fluorography. (B) HeLa-RI₃₃₂ and HeLa- RI₃₃₂-Thr cells, grown in 10-cm dishes, were left untreated (lanes a′ and e′) or preincubated with tunicamycin (5 μg/ml) for the times indicated (lanes b′–d′ and f′–h′). Cell extracts were prepared in Triton X-100–containing buffer, and the pellets were dissolved in an SDS-containing buffer. Cell extracts (20 μg of total protein, corresponding to approximately one-tenth of each extract; lanes a′–d′) and corresponding amounts of the dissolved pellets (lanes e′–h′) were subjected to SDS-PAGE. After transfer of the proteins to nitrocellulose, immunodetection was performed with the use of the anti-BiP antibody and the ECL kit. (C) HeLa-RI₃₃₂ cells, grown in 6-cm dishes, were preincubated and pulse labeled for 30 min in the absence (lanes a" and c") or presence of tunicamycin (5 μg/ml; lanes b" and d"). Anti-BiP immunoprecipitates obtained from cell lysates were subjected to SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane. The membrane was probed by Western blot analysis with the use of anti-BiP antibodies (lanes c" and d"), and after decay of the signal generated by the ECL reaction, the immunoprecipitates were analyzed by autoradiography with the use of BioMax MR x-ray film (lanes a" and b").