L. A. Rutkevich et al. Figure 5. Combined knockdown of PDI and ERp57 substantially impairs protein folding. HepG2 cells constitutively depleting ERp57 by shRNAmir were subjected to knockdown of PDI by siRNA for 6 d. HepG2 cells transfected with control siRNA were prepared in parallel. Pulse-chase experiments were conducted and analyzed as described in Figure 3. (A) Kinetics of disulﬁde formation. (B) Secretion kinetics. Amount secreted was quantiﬁed as a percentage of the total signal recovered at the 10 min chase time point. Black and gray bars represent control and knockdown conditions, respectively. (C) Quantiﬁcation of oxidative folding. Solid lines, control conditions; dashed lines, knockdown conditions. (D) ER to Golgi transport kinetics. (E) Quantiﬁcation of the gels in panel D. Error bars represent the average of three independent experiments one SD, except in the case of TF which was examined in a single experiment. Asterisks indicate quantiﬁcations were not included beyond 30 min of chase due to substrate degradation. proteins was simply masked by the action of PDI or ERp57. Alternatively, ERp72 and P5 may not act on these substrates but are speciﬁc for other client proteins. To distinguish between these possibilities as well as to characterize the degree of functional overlap between PDI and ERp57, PDI family members were depleted in combination. Combined Knockdown of PDI Family Members Initially, PDI and ERp57 were knocked down with high efﬁciency (Figure 1A). As shown in Figure 5, A and B and quantiﬁed in Figure 6A, we observed a marked decrease in the total radioactive signal recovered from cell lysates and medium in double-knockdown cells beyond 30 min of chase, consistent with degradation of all substrate proteins (ex- plored further below). Because this degradation complicated the determination of the percentage of fully oxidized or ER-localized forms of the substrates at later time points, quantiﬁcations were carried out up to the 30-min time point but not beyond (indicated by the asterisks in Figure 5, C and 3100 E). Additionally, the amount of each substrate secreted was quantiﬁed as a percentage of the total radioactive signal recovered at the 10-min chase time rather than as a percent- age of the total signal recovered at each chase time. Figure 5, A and C shows that the oxidative folding of Alb was delayed to the same extent as when PDI alone was depleted, sup- porting our ﬁnding that ERp57 is not acting on this nongly- cosylated substrate (Figure 4). For the glycoprotein sub- strates, a range of impairments in oxidative folding was observed. For FP, there was little change relative to PDI or ERp57 depletion alone. For 2HS, there was a greater delay in oxidative folding compared with that observed with ei- ther of the single knockdowns and, in the case of TF, oxida- tive folding was profoundly impaired. By 30 min of chase, only 15% of TF was fully oxidized compared with 25 and 50% when either PDI or ERp57 were depleted, respectively (compare Figures 3C, 4C, and 5C). Even after 2 h of chase, only small amounts of fully oxidized TF could be observed (Figure 5A). Furthermore, both secretion and ER to Golgi Molecular Biology of the Cell