Biomedical Engineering Reference
In-Depth Information
4. The cells were rapidly placed on ice to halt receptor traffi cking
and then washed three times with fl uorescence-activated cell
sorting (FACS) buffer (PBS with 2 % FBS) immediately before
use for 5 min each on a shaking platform to remove excess
HGF. Cells were detached from the plate surface using 5 mM
EDTA in DPBS, centrifuged and resuspended in FACS buffer
to the fi nal concentration of 1 × 10
6
cells/100
l.
5. Each sample was divided into three wells on 96-well plate and
mixed with 40
ʼ
g/ml of goat
anti-HGFR antibody and incubated for 40 min at 4 °C. After
washing the cells two times with ice-cold FACS buffer, the
cells were incubated with 2.5
ʼ
l of FACS buffer containing 10
ʼ
g/ml R-phycoerythrin donkey
anti-goat IgG secondary antibody in FACS buffer containing
for 30 min on ice.
6. Following two washes in ice-cold FACS buffer, cells were fi xed
overnight in 2 % formaldehyde in PBS at 4 °C or at room tem-
perature for 20 min. After three additional washes with FACS
buffer, cells were analyzed by fl ow cytometry using a BD
FACSCanto™ instrument and FACS DIVA software version
5.0.1 (BD Biosciences). Typically 20,000 cells are acquired for
each time point. To quantify changes in surface MFI, the MFI
of the cells at each time point (tx) following ligand treatment
was compared to untreated cells (MFI
unt
) and calculated using
the following equation: MFI = MFI
tx
- MFI
unt
. An example of
the results with SE is shown in Fig.
2a
.
7. For degradation studies, the transfection mixture of Ube3C-3
siRNA or NT siRNA, DharmaFECT 2, and 1.4 × 10
5
cells in
2 ml of DMEM containing 10 % FBS was plated in individual
wells of a six-well plate and incubated overnight at 37 °C.
8. 24 h later, the transfection mixture was replaced by SF DMEM
and the cells were serum starved for an additional 24 h at 37 °C.
9. 48 h following the transfection at ~70 % confl uency, cells were
pretreated with 1 ml of 10
ʼ
g/ml cycloheximide in SF DMEM
for 1 h at 37 °C to inhibit protein synthesis.
10. Internalization and subsequent degradation of MET was initi-
ated by treating HeLa cells with 100 ng/ml HGF and 10
ʼ
g/
ml cycloheximide in SF DMEM prewarmed to 37 °C for 0, 1,
2, 3, or 4 h. MET degradation was terminated by placing the
plates on ice, washing them with prechilled PBS, and cells were
incubated in TGH lysis buffer containing protease inhibitors
overnight at 4 °C.
11. Protein lysates (40
ʼ
g of protein per lane) were analyzed by
SDS-PAGE and western blot analysis. MET was detected using
40 ng/ml MET c-28 antibody. As a loading control monoclonal
antibody recognizing
ʼ
ʲ
-actin was used at dilution 1:20,000.
