FIGURE 4.

BiP siRNA promotes the ERAD of D18G TTR and amyloidogenic M-TTRs. A and B, 25 nm BiP siRNA (siBiP) or control siRNA (siCtrl) was transiently co-transfected with wild-type TTR (A) or D18G TTR (B) in HeLa cells. After 48 h, cells were labeled with [³⁵S]methionine/cysteine for 15 min and chased at the indicated times. Intracellular and secreted TTRs were immunoprecipitated and loaded onto SDS-polyacrylamide gel, followed by autoradiography (top). The signals for TTR were quantified and plotted (bottom). Secretion efficiency (%) = 100 × ((secreted TTR at given time t)/(intracellular TTR at t = 0 min)). The mean and S.E. values were calculated from two independent experiments. C and D, TTRs (C) or M-TTRs (D) were transiently co-transfected with 25 nm BiP siRNA or control siRNA in HeLa cells. After 42 h, cells were treated with 30 μm proteasome inhibitor MG-132 for 6 h. Cell lysates were immunoprecipitated (IP) with anti-TTR antibody. The immunoprecipitates and cell lysates (Input) were resolved by SDS-PAGE and analyzed by Western blotting with anti-ubiquitinylated protein, anti-TTR, anti-KDEL, and anti-actin antibodies. Ub-TTR and Ub-protein, ubiquitinylated TTR and ubiquitinylated proteins, respectively. WT, wild type.