The cytosolic forms of PrP were targets of Hsp70. A and B. Immunoprecipitation assays. HEK293 cells were co-transfected with pcDNA-PrP-PG5 and pEGFP-Hsp70. Forty-eight hours after the transfection, the cells were harvested, and 400 μg of the whole cell lysate proteins was used in the immunoprecipitation assays. In (A), PrP was immunoprecipitated and blotted with anti-Hsp70. In (B), Hsp70 was immunoprecipitated and blotted with anti-PrP. C. Immunofluorescence. HEK293 cells co-transfected with pEGFP-Hsp70, and various PrP plasmids were stained for 48 h. The individual and merged images of PrP (red), Hsp70 (green), DAPI (blue) were monitored under confocal microscopy. The preparations of the various PrP constructs are indicated on the left. D. The cells infected with various PrP plasmids were extracted subcellular proteomes and examined separately by Western blot analysis using PrP-, Hsp70- and Calnexin-specific antibodies. C: cytosol fraction M: membrane/organelle fraction. E. Immunoprecipitation assays of cytosol fraction. The cytosol fractions extracted from the cells transfected with various PrP plasmids were to be immunoprecipitated by anti-PrP and blotted with anti-Hsp70.