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S. Pédeboscq et al. / European Journal of Medicinal Chemistry 45 (2010) 2473e2479
coverslip was rinsed twice with 1% BSAePBS for 15 min followed by
an additional washing step with PBS. The glass coverslip was dried
and cells were incubated for 1 h with the secondary FITC-conju-
gated goat anti-mouse antibody diluted (1:50) with 1% BSAePBS.
The coverslip was rinsed, dried and mounted in mineral oil on
a microscope slide. Fluorescent images of immunolabeled cells
were observed using an Olympus BH-2 fluorescent microscope and
captured with an Olympus C-5060 digital camera.
appropriate dilutions of tested compounds. Cells were incubated in
Krebs Ringer buffered saline (NaCl 130 mM, KCl 3.6 mM, HEPES
10 mM, NaHCO3 2 mM, NaH2PO4 0.5 mM, MgCl2 0.5 mM, CaCl2
1.5 mM, glucose 4.5 g/l, pH 7.42) supplemented with 200 nM
TMRM and maintained at 37 ꢂC in 5% CO2, 95% air atmosphere for
30 min. Verapamil (20 mM) was added during TMRM incubation to
block multidrug-resistant (MDR) pumps. Cells were then incubated
with trypsin at 37 ꢂC, resuspended in HBSS supplemented with 4%
fetal calf serum, and maintained at 4 ꢂC. 96-well microtiter plates
were then analyzed on a FACSCalibur flow cytometer (Becton
Dickinson) equipped with a 488-nm argon ion laser and fluores-
cence emission was measured in FL3 log mode. Forward scatter
(FSC) and side scatter (SSC) were used to gate out cellular frag-
ments. The number of apoptotic cells was calculated using the Cell
Quest software.
5.2.3. Immunoblot analysis of EGFR
Cells were cultured in 60 mm Petri dishes and incubated with
various concentrations of temozolomide, carboplatin, gefitinib or
bortezomib for 48 h. Cells were lysed at þ4 ꢂC with lysis buffer
(protease inhibitor cocktail (SigmaeAldrich) supplemented with
0.3% SDS and 0.75% b-mercaptoethanol), sonicated and cleared by
centrifugation. Supernatant was collected and protein concentra-
tion determined using the Bio-Rad Protein Assay kit (Bio-Rad
Laboratories GmbH, München, Deutschland). An equal volume of
Acknowledgements
Laemmli solution supplemented with
added to a supernatant aliquot and the mixture was heated for
10 min at 80 ꢂC. Samples (15
g of protein) were loaded on each
b-mercaptoethanol (5%) was
We would like to thank Mrs Chantal Medina for technical
assistance and competence on the FACSCalibur flow cytometer.
m
lane and proteins were separated by 10% SDS polyacrylamide gel
electrophoresis. Proteins were then transferred onto a PVDF (pol-
yvinylidene fluoride) membrane (NEN Life Science Product, Boston,
MA). Membrane was blocked for 2 h in TBST (Tris buffered saline
Tween 20) containing 10% low fat dry milk and probed with
primary antibody diluted in TBST with 5% low fat dry milk over-
night at 4 ꢂC. Primary antibody dilutions were as follows: 1/4000
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