Figure 3.

Association of newly synthesized RBP with ER-chaperones. (A) Posttranslational disulfide oxidation of RBP was carried out as described previously. At chase times of 0, 30, and 90 min, samples were alkylated with 20 mM NEM and split in two. One was treated with 2 mM DSP in DMSO at room temperature for 30 min, whereas the other was mock treated with just DMSO to serve as control. The excess DSP was quenched with 100 mM glycine, microsomes were lysed and subjected to immunoprecipitation with anti-RBP antibody. The immunoprecipitates were analyzed by 13% nonreducing and reducing SDS-PAGE followed by autoradiography. (Left) Nonreduced SDS-PAGE of control and cross-linked samples (lanes 1–6). (Right) Reducing SDS-PAGE of the same samples (lanes 7–12). The positions of the untranslocated (Ut), reduced (Red), folding intermediate (It), and the fully oxidized (Ox) forms are indicated. (B) HepG2 cells were subjected to metabolically labeled, cross-linked and immunoprecipitated with antibodies to RBP, Grp94, BiP, and PDI. The samples were analyzed on reducing, 8–15% gradient SDS-PAGE. (C) Posttranslational oxidation of RBP was carried out as described under Materials and Methods. The samples were lysed with 2% CHAPS, 1× protease inhibitor cocktail, in phosphate-buffered saline, pH 7.2. The lysate was divided into three aliquots and subjected to immunoprecipitation using anti-RBP, anti-BiP, and anti-calnexin antibodies. The immunoprecipitates were analyzed by 13% SDS-PAGE. (D) Quantitation of RBP bands is shown for RBP immunoprecipitated by anti-calnexin (closed squares) and anti-BiP (open squares). The RBP immunoprecipitated by anti-RBP antibody was taken as 100% for respective chase point. (E) RBP was translated in presence of microsomes and cross-linked as described for Figure 3A. Sample corresponding to 0 min was lysed and equal aliquots were immunoprecipitated with antibodies to RBP, Grp94, and BiP. Samples were analyzed by reduced SDS-PAGE.