Figure 3.
Effect of PDI knockdown on oxidative folding of secretory proteins. HepG2 cells were depleted of PDI or treated with control siRNA for 6 d, radiolabeled with [35S]Met for 3 min, then chased with unlabeled Met for the indicated times. The medium was removed, and then cells were treated on ice with 20 mM NEM in PBS and lysed in RIPA buffer containing 20 mM NEM. Both cell lysates and media were immunoisolated with antisera directed against the indicated proteins. (A) Kinetics of disulfide formation. Immune complexes from cell lysates were subjected to SDS-PAGE under reducing (first lane) or nonreducing conditions and proteins were visualized and quantified by phosphorimaging. Shown are representative gels of three independent experiments for substrates albumin (Alb), α-fetoprotein (αFP), transferrin (TF), and α2-HS-glycoprotein (α2HS). Reduced (R), partially oxidized (PO), oxidized (O), and Golgi (G, G1) forms of each protein are indicated. (B) Secretion kinetics. Immune complexes recovered from media samples at the indicated chase times were analyzed by SDS-PAGE and visualized and quantified by phosphorimaging. Histograms indicate the amount of protein observed in the medium as a percentage of the combined intra- and extracellular signal at each chase time. Black and gray bars represent control (C) and knockdown (KD) conditions, respectively. (C) Quantification of gels shown in panels A and B was carried out by expressing the amount of fully oxidized protein in lysate and media samples as a percentage of the total combined amount of reduced, partially oxidized, and oxidized forms at a given time point. Solid lines represent control conditions; dashed lines represent knockdown conditions. (D) ER to Golgi transport kinetics. The indicated proteins were immunoisolated and either digested or not with endo H as indicated and analyzed by reducing SDS-PAGE. Endo HS and Endo HR represent endo H–sensitive and -resistant species, respectively. G and ER represent Golgi-processed and ER forms which could be resolved without endo H digestion. (E) Quantification of the gels in panel D. Histograms represent the amount of protein remaining in the ER as a percentage of the total protein recovered from cells and medium at each time point. Black bars, control; gray bars, knockdown. Error bars represent the average of three independent experiments ± one SD, except in the case of TF which was examined in a single experiment.