Further, GAGs have been reported to act as receptors for PrPSc on the cell surface [26], [27], affect PrPC trafficking [28], [29], [30] and are also found in PrPSc associated plaques [31], [32].
Quantification of PrP expression relative to M17 shows levels of 650, 710, 750, 610, and 5% in PrPC, PrPΔ51 89, PrPΔ23 89, PrP102L, and PrP231stop cells respectively (Fig. 4).
The role of the octarepeat region was investigated in C4/− mice lacking amino acids 32 to 93 of PrPC (30).
Radiolabeling of M17, PrPC, and PrP231stop cells with 59FeCl3-citrate complex for 4 hours shows significantly more 59Fe-ferritin in PrPC cells compared to M17 as above, and minimal change in PrP231stop samples (Fig. 3C, lanes 1 3, black arrow).
As shown in Figure 3, PrPC associates in MVs with the gangliosides GM3 (Fig. 3A) and GM1 (Fig. 3B) and with the tyrosine kinase Fyn (Fig. 3C).
In agreement with the work of Graner [16], PrPC was found to associate with laminin in 1C115-HT and 1C11NE cells (fig. 9C, left panel).
As shown in Figure 1B, PrPC expression in MVs was confirmed by flow cytometry: staining with the anti-PrP polyclonal antibody C-20 PE revealed a specific PrPC reactivity.
To 50µl PrPC substrate (UBH or cell lysate as prepared above), 50 µl of IBH, diluted a further 1/50 in the appropriate buffer supplemented with 1% (v/v) Triton X-100, was added.
Western blotting of M17, PrPC, and PrP231stop lysates and medium sample from PrP231stop cells cultured overnight in serum-free medium with 3F4 shows the expected glycoforms of PrP in PrPC lysates, and undetectable reactivity in M17 and PrP231stop lysates as expected (Fig. 3D, lanes 1 3).
To evaluate if the difference in ferritin iron content of different cell lines is maintained in the presence of excess extra-cellular iron, M17, PrPC, PrPΔ51 89, PrPΔ23 89, PrP102L, and PrP231stop-cells were cultured overnight in the presence of 0.1 mM ferric ammonium citrate (FAC).
To determine if the difference in cellular iron levels between cell lines is due to differential release into the medium, M17, PrPC, PrPΔ51 89, PrPΔ23 89, and PrP102L cells were cultured in the presence of 3H-thymidine overnight to monitor cell proliferation and radiolabeled with 59FeCl3 for 4 hours as above.
