Prion Disease Pathogenesis - Recent Advances in Microbiology - page 58

M17 lysates show barely detectable levels of PrP C , while transfected cell lines express
significantly higher levels of PrP C and mutant PrP forms (Figure 1B, lanes 1-5).

Figure 1. Cells expressing normal and mutant PrP forms incorporate different levels of iron. (A)
Diagrammatic representation of PrPC and mutant PrP forms evaluated in this study. (B) Lysates of
M17, PrP C , PrP Δ 51-89 , PrP Δ 23-89 , and PrP102L were resolved by SDS-PAGE and immunoreacted for PrP
and

-actin. All transfected cell lines express 6-7 fold higher levels of PrP relative to non-transfected
M17 cells (lanes 1-5). (C) Cell lines in (B) were radiolabeled with 59 FeCl 3 -citrate complex, washed
with PBS supplemented with 100 μM DFO to chelate surface bound iron, and lysed. Equal amount of
protein from each sample was spotted on a PVDF membrane, air dried, and exposed to an X-ray film.

β

To evaluate if PrP C or mutant PrP forms infl uence cellular iron uptake, M17, PrP C ,
PrP Δ51-89 , PrP Δ23-89 , and PrP 102L cells cultured in serum-free medium for 1 hr were radio-
labeled with 59 FeCl 3 -citrate complex for 4 hr in the same medium, washed with PBS
supplemented with 100 μM desferrioxamine (DFO) to remove surface bound iron,
and lysed in non-denaturing buffer. Equal amount of protein from lysates was spotted
on a PVDF membrane, air-dried, and exposed to an X-ray fi lm. Surprisingly, PrP C and
PrP 102L cells incorporate signifi cantly more 59 Fe, while PrP Δ51-89 , PrP Δ23-89 cells take up
less 59 Fe than M17 controls (Figure 1C).