FIGURE 7:

Overexpression of ERp57 or ERp72 leads to an increase in ApoB100 ERAD. (A) McArdle-RH7777 cells were transfected with pcDNA3.1 lacking an insert (–) or containing human (h) PDI, ERp57, or ERp72. Equal amounts of cell lysates were analyzed by immunoblotting with an anti-PDI antibody, anti-ERp57 antibody, or anti-ERp72 antibody. (B) Following a metabolic labeling reaction, a 60-min chase was performed as described in Materials and Methods in McArdle-RH7777 cells transfected with a vector control (pcDNA3.1 lacking an insert) or containing the PDI, ERp57, or ERp72 genes. The bars indicating “Relative Recovery of ApoB” indicate the amount of ApoB-precipitable material recovered from cell lysates and secreted into the medium at the completion of the chase divided by the amount of ApoB-precipitable material recovered after 15 min of chase (this prolonged period is required to complete the synthesis of ApoB (Gusarova et al., 2001)). Data represent the means of three experiments, ± SEM. *p < 0.05, **p < 0.01. (C) Pulse-chase reactions were performed as in B, except that either DMSO or the proteasome inhibitor MG132 was added to the cells prior to the start of the metabolic labeling. Data represent the means of three experiments, ± SEM. *p < 0.05.