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and secretion of PrP
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in transfected cells (Figure 3D, lane 4) [22, 23]. Re-probing
of lysate samples for ferritin, Tf, and TfR shows increased levels of ferritin and de-
creased levels of Tf and TfR in PrP
C
samples compared to M17 lysates (Figure 3E,
lanes 1 and 2). The PrP
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lysates show minimal change in ferritin levels, and sur-
prisingly, lower levels of Tf and TfR relative to M17 lysates (Figure 3E, lanes 1 and
3). This observation is surprising since
59
Fe-ferritin levels in PrP
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cells are as low
as M17, and yet the cells do not show increased levels of Tf and TfR as in M17-cells.
Reaction for β-actin confi rms equal loading of protein in all samples (Figure 3E, lanes
1-3).
Figure 3.
Expression of PrP on the plasma membrane is essential for iron incorporation in ferritin.
(A)
59
Fe-labeled M17 and PrP
C
lysates were fractionated on a 3-9% native gradient gel and auto-
radiographed (lanes 1 and 2), or immunoblotted as above with antibodies specific to PrP, ferritin, TfR,
and Tf (lanes 3-10). (B) Immunoblotting of the same samples following fractionation by SDS-PAGE
shows similar differences in the levels of PrP, ferritin, Tf, and TfR as in (A) after normalization with
actin (lanes 1 and 2). (C)
59
Fe-labeled M17, PrP
C
, and PrP
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lysates were fractionated by native gel
electrophoresis and subjected to autoradiography (lanes 1-3). (D) Unlabeled lysates prepared from
M17, PrP
C
, and PrP
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lysates, and methanol precipitated proteins from the medium sample of
PrP
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cells were fractionated by SDS-PAGE and immunoblotted for PrP using 3F4 (lanes 1-4). (E)
Membrane from (D) was re-probed for ferritin, Tf, TfR, and
β
-actin (lanes 1-3).