Figure 1
DE resistant CWD and sheep scrapie are transported across Caco2 monolayers. (A) CWD and sheep scrapie brain homogenates were treated with DE and subjected to immunoblotting. Probing for PrP reveals DE-resistant PrP forms in all four cases of CWD and sheep scrapie (lanes 1-8). (Lanes 1, 3, and 5 were loaded with 5 μl of each sample, lanes 2, 4, and 6 with 10 μl of the same sample, and lanes 7 and 8 with 15 μl of sample to confirm efficient cleavage of PrPSc). (B) Control and DE treated NH, CJDH, and CDW-4 samples were immunoprecipitated with anti-ferritin antibody and antibody-protein complexes eluted from beads and unbound proteins in the supernatant were subjected to Western blotting. Probing for PrP shows significant co-immunoprecipitation of PrP with ferritin from DE+ CJDH and CWD-4 samples (lanes 8 and 12). No PrP is detected in DE+ NH samples as expected (lanes 3 and 4, *) and in the supernatant of DE+ CJDH and CWD-4 samples (lanes 7 and 11). Significant amounts of normal PrP and full-length PrPSc co-immunoprecipitate with ferritin from DE- NH, CJDH, and CWD-4 samples (lanes 2, 6 and 10). (C) Model of a trans-well demonstrating the separation of AP and BL chambers by a monolayer of Caco-2 cells. (D) DE treated brain homogenates from sheep, CWD-1, and CWD-2 were re-suspended in PBS and added to the AP chamber of a tight monolayer of Caco-2 cells. After an overnight incubation, proteins from AP and BL chambers were precipitated and subjected to Western blotting. Probing for PrP shows significant transport of DE resistant PrPSc from sheep, CWD-1 and CWD-2 samples to the BL chamber (lanes 1-6). Re-probing for ferritin shows similar transport of ferritin from AP to the BL chamber (lanes 1-6). (E) Immunostaining of filter inserts for the tight junction protein ZO-1 shows an intact monolayer of Caco-2 cells with tight junctions (panels 1 and 2). Bar: 10 μm.