Figure 8.

RI₃₃₂ and RI₃₃₂-Thr are readily cross-linked to BiP in tunicamycin-treated cells. HeLa-RI₃₃₂ (A, lanes a–d, and B) and HeLa-RI₃₃₂-Thr (A, lanes e–h, and C) cells were pretreated and pulse labeled for 30 min in the absence (A) or in the presence (B and C) of tunicamycin (Tu; 5 μg/ml). Cells were lysed in the presence of digitonin (0.2%) and incubated in the absence or presence of DSP (100 μg/ml) as indicated. As a control, a cross-linking experiment was carried out on a cell lysate preincubated with ATP (5 mM; B, lanes e′ and f′; C, lanes e" and f"). Ribophorin I (RI) and RI₃₃₂ or RI₃₃₂-Thr were immunoprecipitated from the cell lysates in the presence of SDS and Triton X-100 (A, lanes c, d, g, and h; B, lanes c′–e′; C, lanes c"–e"). Anti-BiP immunoprecipitations were performed under the same conditions (A, lanes a, b, e, and f; B, lanes a′, b′, and f′; C, lanes a", b", and f"). All samples were analyzed by SDS-PAGE followed by fluorography. Exposure times: A, 7 d; B, lanes a′, b′, and f′, 2 d; lanes c′–e′, 7 d; C, lanes a", b", and f", 1 d; lanes c"–e", 7 d.