FIGURE 6.
Overexpression of ER quality control molecules reverses formation of PrPc aggregates and PrPSc accumulation. A, N2a cells were transiently transfected with EDEM-3 (lanes 3, 4, 7, and 8) or ERGIC-53 (lanes 11, 12, 15, and 16). 16 h before harvesting, cells were treated with tunicamycin (T) or DTT (D) to induce ER stress and subsequently subjected to solubility assay. Immunoblots show the amount of PrP collected in soluble and insoluble fractions probed with mAb 4H11. Significantly less aggregated PrP was found in cells overexpressing ERGIC-53 or EDEM-3. the lowest panels confirm successful transfection with the indicated constructs. B, prion-infected L929-22L cells transiently overexpressing EDEM-3 or ERGIC-53 were harvested 72 h post transfection and subjected to PK digestion to isolate PrPSc. Upon SDS-PAGE, amounts of PrPSc were analyzed by immunoblot. All cells overexpressing transfected proteins contain less PrPSc than mock transfected ones (lanes 3 and 4, and 7 and 8). Expression of quality control molecules is shown in the lowest panels with specific antibodies. C, evaluation of two independent experiments performed in triplicate confirms significant reduction of PrPSc in prion-infected cells overexpressing EDEM-3 or ERGIC-53.