Quantitative Measurement of the Activation of Signaling Pathways Using
Two-Color Infrared Fluorescent Western Blotting and Cell-Based Assays
Huaxian Chen and D. Michael Olive
LI-COR, Inc., 4308 Progressive Avenue, Lincoln, NE 68504
[email protected]
Two-Color Western Blots
Abstract
There are more than 500 protein kinases and 100 protein phosphostases encoded in
the human genome. Protein phosphorylation/dephosphorylation by these kinases
and phosphotases is a critical process regulating almost every signal transduction
event. The study of protein phosphorylation has largely relied on conventional
Western blot and enzymatic kinase assays. Since both assays can assess only one
target at a time, an additional step is usually needed for data normalization.
Two-Color Cell Based Assays
IFN-γγ Induced Tyrosine Phosphorylation of IFN-γγ Receptor in Hela Cells
700 nm (red), 800 nm (green), both (yellow)
Quantitative and simultaneous measurements of EGFR and phosphorylation
of EGFR in response to EGF in the presence of EGFR inhibitor
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700 nm alone
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3
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Inhibitor
EGF
800 nm alone
-
+
3
+
µm
1.5
+
750
+
375
+
180
+
nm
90
+
Inhibition of EGFR Phosphorylation
by P168393
45
+
22
+
11
+
5.5
+
Color image
Black/white
Furthermore, we have extended the assessment of the phosphorylation state of the
EGF receptor using simultaneous direct two-color immunostaining of stimulated and
unstimulated cells. Dose-response curves demonstrated that the quantitative cellbased assay is sensitive, highly reproducible and linear over a wide dynamic range.
The ability to monitor the activation of signaling pathways by in vitro and in situ
assays should greatly facilitate the characterization of pharmacological inhibitors and
target validation of specific molecules within the cell.
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700/800 nm
120
% of Phosphorylation
We have developed methods employing two-color infrared fluorescent technology
for the analysis of signal transduction events. With three different systems (INF-γ,
ERK1/2, and Stat3), we have demonstrated the capability of two-color Western blots
to simultaneously detect both the phosphorylated protein and the total protein
regardless of its phosphorylation status.
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A431 cells were seeded in a 96-well plate and grown to be confluent. The cells were serum-starved overnight. Twofold serial dilutions of the EGFR inhibitor, PD168393, were added to wells 3 through 12, and were incubated at 37°C
for 1 hour. The cells in wells 2 to 12 were stimulated with 100 ng/ml of EGF for 15 minutes. After stimulation, the
cells were fixed with 4% formaldehyde/PBS for 20 minutes at room temperature and washed three times with 0.1%
Triton X-100/PBS. After blocking, the total EFGR (visible in green) and phosphorylated EGFR (visible in red) were
revealed by incubation with rabbit α-EGFR and mouse α-phosphorylated EGFR, followed by incubation with Alexa
Fluor® 680-labeled goat α-mouse and IRDye 800-labeled goat α-rabbit antibodies.
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Number of Cells (x10-6 )
Tyrosine Phosphorylation of ERK in Mast Cells
Induced with Multivalent Antigens
Through Receptor-Bound IgE
Quantitative and simultaneous measurements of total ERK and
phosphorylation of ERK in response to EGF in the presence of EGFR inhibitor
EGF Induced Tyrosine Phosphorylation
Of Transcriptional Factor Stat3
700 nm (red), 800 nm (green), both (yellow)
Inhibitor
EGF
700 nm (red), 800 nm (green), both (yellow)
1 2
3
-
+
3
+
µm
1.5
+
750 375
+
+
180
+
nm
90 45
+
+
22
+
11
+
800 nm alone
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700 nm alone
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3 4
5
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800 nm alone
800 nm (total ERK)
ERK Phosphorylation As the Result
Of Inhibition of EGFR by P168393
5.5
+
700/800 nm
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700 nm alone
Primary antibodies against INF-γR, EGFR, ERK, Stat3 and their phosphorylated forms
(4G10, phospho-EGFR, phospho-ERK, phospho-stat3) were purchased from commercial sources. Goat anti-mouse and anti-rabbit secondary antibodies were labeled
with IRDye 38 (800 nm, Rockland Immunochemicals) or with Alexa Fluor® 680 (700
nm, Molecular Probes).
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3
2
Resting
INF-gamma
150
IFN-γ receptor was immunoprecipitated from resting (lanes 1 to 4) and IFN-γ treated (lanes 5
to 8) cells. Two-fold serial dilutions of the immunoprecipitated proteins were separated by
SDS-PAGE, transferred onto a nitrocellulose membrane, and detected simultaneously with
mouse anti-phosphotyrosine (visible in red) and rabbit anti-IFN-γ receptor (visible in green).
The yellow color in the top panel indicates the presence of both dyes in a given pixel.
Materials & Methods
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Log of Inhibitor Concentration (1=10 nM)
200
Number of Cells (x10-6 )
LI-COR has developed an IR imaging system designed to image membranes and
plates for protein application. The imager simultaneously detects two distinct wavelengths. A scanning optical assembly carries two laser diodes that generate excitation
light at 680 and 780 nm, as well as two avalanche photodiodes, which detect emitted fluorescence at 720 and 820 nm. Here we demonstrate two-color infrared fluorescent technology for the analysis of signal transduction events by in vitro and in situ
assays.
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700 nm (phosphorylated EGFR)
120
% of Phosphorylation
Intensity
Intensity
15
0
Common visible fluorophores cannot be used effectively for direct protein detection
on membranes and in plastic plates because of their high background fluorescence
in the visible range. Near-infrared (IR) fluorophores (670-1100 nm) have a distinct
advantage over visible dyes, in that very low background fluorescence at longer
wavelengths provides an excellent signal-to-noise ratio. Furthermore, antibodies
labeled with IR dyes at different wavelengths can be used for detection of multiple
targets on membranes and in plates, a feature that cannot be accomplished by other
technology such as ECL and radioisotopes.
800 nm (total EGFR)
250
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Introduction
The Amount of IFN-γR Immunoprecipitated
From Resting and INF-γ Treated Cells
Quantification of IFN-γ Stimulated Tyrosine
Phosphorylation of INF-γR
700 nm (phosphorylated ERK)
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80
60
40
20
0
0
- 20
1
2
3
Log of Inhibitor Concentration (1=10 nM)
Color image
Color image
A431 cells were treated as described above. The total ERK (visible in green) and phosphorylated EGFR (visible in red)
were revealed by incubation with rabbit α-ERK and mouse α-phosphorylated ERK, followed by incubation with Alexa
Fluor® 680-labeled goat α-mouse and IRDye 800-labeled goat α-rabbit antibodies.
The basic procedure for two-color infrared fluorescence detection is as follows:
Black/white
Black/white
Mouse antibody against the phosphorylated protein
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80
60
40
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IRDye 38-labeled goat α-rabbit (green)
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0
5
10
15
Number of Cells (x10 -4 )
Odyssey™ Analysis
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The Amount of Total ERK Loaded from Resting
And Antigen Activated Cells
Intensity
Intensity
100
Alexa Fluor® 680-labeled goat α-mouse (red)
1 2 3 4 5 6 7 8
y
Quantification of
Antigen-stimulated Tyrosine
Phosphorylation of ERK
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100
90
80
70
60
50
40
30
20
10
0
1 2
120
Quantification of EGF-Stimulated
Tyrosine Phosphorylation of
Stat3 in A431 Cells
100
Resting
Ag Activated
2 3 4 5 6 7 8
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60
20
5
10
15
20
25
Number of Cells (x10-4 )
Mast cells sensitized with anti-DNP IgE were reacted with
DNP38-albumin for 5 minutes. Two-fold serial dilutions of total
cellular lysate from resting (lanes 1 to 4) and activated (lanes 5 to
8) cells were separated by SDS-PAGE, transferred onto nitrocellulose membrane, and detected simultaneously with mouse antibodies recognizing only tyrosine-phosphorylated ERK (visible in
red), and rabbit antibodies recognizing ERK regardless of its
phosphorylation status (visible in green).
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60
40
Resting
EGF-Treated
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0
0
0
5
10
15
Number of Cells(x10 -4)
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25
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Conclusions
• Two-color Western blot and in situ assays allow two antigens to be analyzed simultaneously on the same
membrane or in the same well, a feature that is critical for drug screening, yet which cannot be offered by
other technologies such as ECL and radioisotopes.
The Amount of Total Stat3
Loaded From Resting and
EGF-Treated Cells
100
40
0
3 4 5 6 7 8
Phosphorylation of Stat3 in A431 Cells
120
Intensity
p
Simultaneously added to Western blots or plates
Intensity
1 2 3 4 5 6 7 8
Rabbit antibody against the total protein regardless of its phosphorylation status
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5
10
15
20
25
30
Number of Cells (x10 -4)
A431 cells were stimulated with EGF for 10 minutes. Two-fold
serial dilutions of total cellular lysate from resting (lanes 1 to 4)
and activated (lanes 5 to 8) cells were separated in SDS-PAGE,
transferred onto nitrocellulose membrane, and detected simultaneously with mouse antibodies recognizing only tyrosine-phosphorylated Stat3 (visible in red), and rabbit antibodies recognizing Stat3 regardless of its phosphorylation status (visible in green).
• Quantitative measurements are linear over a wide range and the fluorescent signals are directly proportional
to the amount of antigens present on membranes and in wells without the influence of exposure time and
stripping as in ECL. Infrared fluorescent analysis offers an excellent technique enabling the large-scale
analysis of signal transduction, including drug screening.
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Application Note #549
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