Biomedical Engineering Reference
In-Depth Information
protease treatments. To enhance cell attachment, plated cells were
cooled on ice for 5 min prior to media changes. Immediately
prior to transfections, HeLa cells were harvested and resus-
pended in DMEM supplemented with 10 % fetal bovine serum
(FBS) at a concentration of 5,000 cells/50
ʼ
l/well of the
2. Recombinant human HGF: HGF (PeproTech; cat#100-39,
Lot# 0109S201) was received as a lyophilized powder and was
reconstituted in water to a concentration of 500
96-well plate (
see
Subheading
3.1
).
g/ml, ali-
quoted and stored at −20 °C. For MET internalization, this
solution was thawed and added to the cell media at a fi nal
concentration of 100 ng/ml.
3. Dynasore: GTPase inhibitor Dynasore was received as a lyoph-
ilized powder from Sigma-Aldrich (cat# D7693) and was
reconstituted in DMSO to a concentration of 30 mM, ali-
quoted and stored at −80 °C. For MET internalization studies,
the Dynasore stock solution was thawed and added directly to
the cell media at a fi nal concentration of 50
ʼ
M.
4. Cycloheximide: Cycloheximide (Sigma-Aldrich; C-7698) was
received as a lyophilized powder and was reconstituted in water
to a concentration of 10 mg/ml. To inhibit protein synthesis
the stock solution was thawed and added to serum-free (SF)
DMEM at a fi nal concentration of 10
ʼ
ʼ
g/ml.
1. DharmaFECT 2 transfection reagent: Transfection reagent
DharmaFECT 2 (Thermo Fisher Scientifi c; cat# I-2002, Lot#
090917T) was used to deliver siRNA into cultured cells and in
our hands gave the highest transfection effi ciency with the low-
est toxicity compared to DharmaFECT 1, 3, or 4 reagents. The
transfection method was optimized using HeLa cells stably
expressing a fi refl y luciferase transgene [
21
] and transfecting
different numbers of cells with differing amounts of siRNA and
over time. The best transfection effi ciency was achieved using
5,000 cells/well in a 96-well plate and 0.1
2.2 Cell
Transfections
and Analysis
l of DharmaFECT
reagent with 40 pmol of siRNA combined with 9.9
ʼ
l serum-
free (SF) media after 48 h. For larger transfection volumes,
DharmaFECT reagent amounts were scaled accordingly.
ʼ
2. Antibodies: Normal goat IgG (Santa Cruz; sc-2028) and goat
anti-human MET (R&D, AF276) were used at 0.2
ʼ
g/ml for
surface MET cell-based ELISA or 10
g/ml for fl ow cytometry
analysis. Horseradish peroxidase (HRP)-conjugated bovine
anti-goat IgG (Jackson ImmunoResearch Laboratories; 805-
035-180) and R-phycoerythrin donkey anti-goat IgG antibody
(Jackson ImmunoResearch Laboratories; 705-116-147) were
used as secondary antibodies. For western blot analysis, MET
antibody c-28 (Santa Cruz, sc-161) was used at 1:5,000,
Ube3C antibody (GeneTex; GTX119102) at 1:10,000, and
ʼ
