Biology Reference
In-Depth Information
Figure 8.
Cross-linking of PrP has minimal effect on ferritin iron content. (A) PrP
C
-cells exposed to 12
μg/ml of 3F4 for 4 hr were radiolabeled with
59
FeCl
3
in the last 2 hr, and lysates were fractionated on a
native gel followed by autoradiography (lanes 1 and 2). Equal aliquots of lysates were fractionated by
SDS-PAGE as above and immunoblotted with 3F4 (lanes 3 and 4). The membrane was re-pobed for
ferritin, Tf, TfR, and
-actin (lanes 5 and 6). (B) Quantification by densitometry shows an increase in
ferritin iron, ferritin, and TfR levels, and insignificant change in Tf levels by 3F4 treatment. *p<0.001,
**p<0.025. n = 3. (C) Estimation of LIP after exposing the cells to 12 μg/ml of 3F4 or anti-Thy-1
antibody for 4 hr shows an increase in 3F4 exposed cells, and a decrease in anti-Thy-1 treated cells.
*p<0.001. n = 7.
β
Prp Does Not Modulate Release of Iron From Cells
To determine if the difference in cellular iron levels between cell lines is due to dif-
ferential release into the medium, M17, PrP
C
, PrP
Δ51-89
, PrP
Δ23-89
, and PrP
102L
cells were
cultured in the presence of 3H-thymidine overnight to monitor cell proliferation and
radiolabeled with
59
FeCl
3
for 4 hr as above. Labeled cells were washed with PBS con-
taining 100 μM DFO to remove surface bound
59
Fe, and chased in complete medium
for 30 min to 16 hr. At the indicated times equal aliquots of medium were retrieved and
released
59
Fe was quantified in a γ-counter. Kinetic analysis shows minimal difference
