Ubiquitination and proteasome-mediated degradation. A. RNA inference. siRNAs for both Hsp70 and negative control RNA were used. HEK293 cells were co-transfected with different siRNA and the plasmid expressing PG14-PrP or Cyto-PrP and harvested 48 h post-transfection. The cell lysates were examined by PrP- and Hsp70-specific Western blots. B. Proteasome effect. HEK293 cells were transiently transfected with the plasmid PG14-PrP or Cyto-PrP. 48 h later, 1 μM GA (final concentration) was added into the culture medium and maintained for another 24 h and 30 μM MG132 (final concentration) was added at the last 6 h. The levels of PrP and actin in the cell lysates were evaluated by individual Western blots. C. Ubiquitination. HA-ubiquitin and various PrPs were transiently expressed in HEK293 cells in the presence or absence of 1 μM GA. Cells lysates were employed into immunoprecipitation captured with anti-PrP antibody. Ubiquitinated proteins were evaluated by ubiquitin specific Western blots.