Figure 6.

Misfolded transferrin is degraded by the proteasome in PDI/ERp57-depleted cells. (A) Pulse-chase data from Figures 3–5 was analyzed for changes in the total amount of radiolabeled Alb, αFP, TF, α₂HS, and α₁AT recovered from the combined intracellular and medium fractions. Total protein, as measured by densitometry, was defined as 100% at the 10 min chase time. Error bars represent the average of three independent experiments ± one SD. (B, C) HepG2 cells were treated with control siRNA or depleted of PDI and ERp57 for 6 d and then incubated in the absence or presence of 5 μg/ml lactacystin for 5 h. Cells were either plated on Cell-Tak-treated coverslips (BD Biosciences), fixed in paraformaldehyde, and permeabilized for confocal microscopy (B) or lysed in RIPA buffer for Western blot analysis (C). Immunoblot or immunofluorescence was carried out using the indicated antibodies (Scale bar = 10 μm).