Biology Reference
In-Depth Information
as described in previous reports [41, 42]. For this study M17 cells from two different
sources were transfected at least three separate times and bulk transfected cells were
used to avoid cloning artifacts. Similarly transfected cells from two different inves-
tigators and cells cultured in DMEM supplemented with 10% FBS and Opti-MEM
supplemented with different lots of FCS were also tried to avoid errors due to culture
conditions.
Radiolabeling with
59
FeCl
3
The M17, PrP
C
, and mutant PrP
Δ51-89
, PrP
Δ23-89
, PrP
102L
, and PrP
231stop
cells cultured
overnight to 80% confluency were serum starved for 1 hr and incubated with
59
FeCl
3
-
citrate complex (1 mM sodium citrate and 20-25 μCi of
59
FeCl
3
in serum free Opti-
MEM; molar ratio of citrate to iron was maintained at 100:1) for 4 hr at 37°C in the
incubator. At the end of the incubation cells were washed 3 times with ice cold PBS
and lysed with native lysis buffer (0.14 M NaCl, 0.1 M HEPES, pH 7.4, 1.5% Triton
X-100 and 1 mM PMSF). Aliquots of lysates were mixed with glycerol (to a final
concentration of 5%) and traces of bromophenol blue, and equal amount of protein
from each sample was resolved on 3-9% native gradient gel. For fractionation on
SDS-PAGE, the same samples were mixed with 4× SDS-sample buffer, boiled for 10
min and resolved on SDS-PAGE followed by immunoblotting.
Native Gradient Gel Electrophoresis, Autoradiography, Immunoblotting, and
Electroelution
Electrophoresis of lysates was performed using a Hoefer SE 600 vertical apparatus
with a cooling system. Linear 3-20% (Figure 1) or 3-9% (Figure 3) gradient poly-
acrylamide gels were prepared as described by Vyoral et al. [21] with modifications.
The gel mixture contained 0.375 M Tris, pH 6.8, 1.5% Triton X-100, and 1.18 mM
ammonium persulfate. N,N,N′,N′-Tetramethylethylenediamine (TEMED) was added
to a final concentration of 5.38 mM. Radiolabeled lysates mixed with glycerol were
subjected to electrophoresis using electrode/running buffer (25 mM Tris, 192 mM
glycine pH 8.3, and 1.5% Triton X-100) under constant current (100 mA) for 4 hr at
4°C. Gels were either electroblotted or vacuum dried (BioRad) and exposed to X-ray
film (Kodak BioMax XAR) fitted with intensifying screens. For Western Blotting,
gels were washed thoroughly with electrode buffer without Triton X-100 for 2 hr (each
wash of 200 ml, 10 min) on a slowly rocking platform to remove Triton. The gel was
electroblotted to a PVDF membrane using BioRad semi-dry electroblotting system
with anode buffer (25 mM Tris, pH 10.4) and cathode buffer (25 mM Tris, 39 mM
glycine, pH 9.2) at 25 V for 90 min. Membranes were further processed for immu-
nodetection as described below. To confirm the identity of iron labeled proteins, iron
bands were excised from native gels and proteins were electro-eluted using Biorad
electro-eluter at 60 mA for 4 hr. Eluted proteins were concentrated by methanol pre-
cipitation and analyzed by SDS-PAGE.
SDS-PAGE and Western Blotting
Cells cultured under different conditions were fractionated by SDS-PAGE and im-
munoblotted as described previously [41, 42]. The following antibody dilutions were
