Figure 10.

RI₃₃₂ and RI₃₃₂-Thr are stabilized after treatment of cells with A23187 and are coimmunoprecipitated with BiP only in the presence of the drug. (A) HeLa-RI₃₃₂ and HeLa-RI₃₃₂-Thr cells were left untreated (lanes a–d) or preincubated with A23187 (5 μM) (lanes e–h). The cells were pulse labeled for 10 min and chased for up to 2 h in the continued absence or presence of the drug. Anti-ribophorin I immunoprecipitations performed on cell lysates were analyzed by SDS-PAGE and fluorography. (B) HeLa-RI₃₃₂ (lanes a′–c′) and HeLa-RI₃₃₂-Thr (lanes d′–f′) cells were left untreated (lanes a′ and d′) or preincubated with A23187 for 30 min (lanes b′ and e′) or 90 min (lanes c′ and f′). Cells were pulse labeled in the continued absence or presence of the drug for 30 min. Cell lysis and immunoprecipitations with the monoclonal anti-BiP antibody were performed as described in MATERIALS AND METHODS. (C) HeLa-RI₃₃₂ (lanes a"–c") and HeLa-RI₃₃₂-Thr (lanes d"–f") cells were preincubated in the absence (lanes a" and d") or in the presence of A23187 (lanes b", c", e", and f") and pulse labeled for 30 min in the continuous absence or presence of the drug. Cell lysis, a 30-min incubation in the absence (lanes a", b", d", and e") or presence (lanes c" and f") of ATP (5 mM), and anti-BiP immunoprecipitations were performed as described in MATERIALS AND METHODS. The anti-BiP immunoprecipitates were eluted from the protein A–Sepharose beads with a buffer containing SDS (2%) and used for a second round of anti-ribophorin I immunoprecipitations under stringent conditions. All samples were analyzed by SDS-PAGE and fluorography. Exposure times: A and B, 2 d; C, 42 d.