Abstract

1
Abstract
Intracellular Analysis of Activated Cell
Signaling Molecules Through the Use of
Phospho-Specific Antibodies and Flow
Cytometry P.J. Burkett, B.J. Nelson, C.L.
Pennington, A. Claxon, M.T. Wilson, J. Apgar, A.
Agadir, L. Stein, A. Stall, R. Campos-Gonzalez,
and J.W. Brooks
Abstract Cell signaling protein
phosphorylation is key to effective transduction
of extracellular signals into the cell to solicit
the desired biological response. Traditionally,
such events have been examined through kinase
assays, phosphoamino acid labeling, and
immunoprecipitation(IP)/ Western blotting (WB).
Phospho-specific antibodies (Abs) have further
enabled the utilization of WB to examine these
changes. We have developed phospho-specific Abs
that can be used to analyze phosphorylation
events by multiple techniques, including Flow
Cytometry (FC). In this study, we have utilized a
general phosphotyrosine monoclonal Ab (mAb) and
mAbs that distinguish between the phosphorylated
and non-phosphorylated forms of Caveolin-1,
Stat5, and Stat1. For FC analysis, cells were
treated, fixed and permeabilized, and incubated
with primary Ab. They were then washed and
stained with anti-mouse IgG-FITC and analyzed on
a BD FACSCalibur. While non-phospho-specific Abs
exhibited similar fluorescence intensity between
resting and activated cells, phospho-specific Abs
exhibited higher fluorescence intensity in
activated cells. FC results correlated with WB
and Immunofluorescence (IF) data. We also
utilized the BD Cytometric Bead Array (CBA)
system to evaluate the phosphorylation state of
Stat1 in denatured lysates from cells expressing
either a functional or nonfunctional Epidermal
Growth Factor (EGF) receptor. While cells
expressing the functional receptor exhibited
EGF-induced phosphorylation of Stat1, cells
expressing the nonfunctional receptor lacked any
increase in Stat1 phosphorylation. In conclusion,
we have identified multiple phospho-specific mAbs
that detect phosphoproteins through intracellular
staining by FC. This application can be employed
in multiparameter analysis to dissect cell
signaling events in a manner that is more rapid,
sensitive, and quantitative than traditional
techniques, such as WB.
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Introduction
Intracellular Analysis of Activated Cell
Signaling Molecules Through the Use of
Phospho-Specific Antibodies and Flow Cytometry
Introduction Intracellular signaling involves a
multitude of pathways and components that are
coordinated for the desired biological response
in the cell. Phosphorylation of Ser/Thr and/or
Tyr residues is often used to regulate activity
or proteinprotein interactions. Phosphoproteins
play central roles in critical cellular decision
processes, including apoptosis, growth control,
cell cycle, cytoskeletal rearrangement, and
transcription. Although the detection of
phosphoproteins has traditionally been examined
through techniques such as kinas assays and
phosphoamino acid labeling, the development of
phospho-specific Abs has allowed for the
widespread use of Western blotting (WB). One of
the initial phospho-specific Abs was the
phosphotyrosine specific PY20. Subsequently, Abs
against specific phosphorylation sites have
advanced the understanding of phosphoprotein
function. However, limitations exist in the time
required to perform WB, the required sample size,
the inability to do multiparameter analysis of a
single sample, and the lack of quantitative
results. In contrast, flow cytometry (FC) allows
for rapid, quantitative, multiparameter analyses
on single cells. Here, we have utilized
phospho-specific mAbs and FC to analyze the
phosphorylation state of intracellular signaling
molecules at the single cell level. In addition
to analyzing general tyrosine phosphorylation
with PY20, we have examined the phosphorylation
of Caveolin-1 and Stat proteins. Caveolins are
trans-membrane adaptor proteins that recognize
GPI-linked proteins, interact with downstream
cytoplasmic signaling molecules, and are involved
in the regulation of the ERK1/ERK2 MAP kinase
signaling pathways. Caveolin-1 is phosphorylated
by Src kinases, primarily at Tyrosine 14.
Phosphorylation is also central to the Jak/Stat
signaling pathway. Stat proteins are latent
cytoplasmic transcription factors. They associate
with active, phosphorylated cytokine and growth
factor receptors and are themselves
phosphorylated by receptor-associated kinases.
Once phosphorylated and
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Intro - cont.
Intracellular Analysis of Activated Cell
Signaling Molecules Through the Use of
Phospho-Specific Antibodies and Flow Cytometry
Introduction (continued) activated, the Stat
proteins dimerize and translocate to the nucleus,
where they bind DNA and induce target gene
expression. Like other Stat proteins, Stat5
functions downstream of multiple cytokine
receptors, including those for IL-2, IL-7, and
GMCSF. Phosphorylation of Stat5 at Tyr694
promotes its binding to the b-casein gene
promoter and is important in the lactogenic
hormone response. On the other hand, Stat1 is
unique in that its function is only vital for the
actions of the Type I and Type II IFNs, although
other cytokines can induce its activation.
Phosphorylation of Stat1 at Tyr701 promotes its
interaction with and modulation of the
transcriptional activity of the ISGF3
(Interferon-Stimulated Gene Factor 3) complex. We
have extended our flow analysis by utilizing the
BD CBA system to evaluate the phosphorylation
state of Stat1 in response to Epidermal Growth
Factor (EGF) stimulation in lysates of cells
transfected with a wild type (WT) or functionally
inactive mutant EGF receptor. The CBA system
employs the sensitivity of amplified fluorescence
detection by FC to measure soluble analytes in a
particle-based immunoassay. The particle is
coated with a capture Ab, analogous to a coated
well in an ELISA plate, and is incubated in
suspension with a small volume sample. The beads
are then probed with a fluorescent-tagged
detection Ab and analyzed by FC. Our results
demonstrate that intracellular phosphoproteins
can be effectively evaluated by FC at the single
cell level and in whole cell lysates. This
approach allows for the analysis of cell
signaling pathway components with technology that
is more rapid, sensitive, and quantitative than
WB. The FC based analysis of cell signaling
pathway activation, in combination with cell
surface markers, allows for the multiparameter
dissection of biologically relevant cell
subpopulations based on the activation state of
pathway components.
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Methods - Cell Culture Immunoblotting
Intracellular Analysis of Activated Cell
Signaling Molecules Through the Use of
Phospho-Specific Antibodies and Flow Cytometry
Methods Cell Culture and Reagents Human
endothelial cells were maintained in DMEM
containing 5 FBS, 5 Calf Serum, and
antibiotics. Following overnight serum
starvation, cells were treated with sodium
pervanadate (2mM) for 20 minutes at 370C. A431
cells were maintained in DMEM containing 10 FBS
and antibiotics. Following overnight serum
starvation, cells were stimulated with EGF (100
ng/ml) for 5 minutes or treated with sodium
pervanadate for 20 minutes at 370C. U937 cells
were maintained in RPMI containing 10 FBS and
antibiotics. Following overnight serum
starvation, cells were stimulated with either
IFNg (1000 U/ml) for 15 minutes or GMCSF (15
ng/ml) for 20 minutes at 370C. B82L mouse
fibroblasts, which lack endogenous EGF receptor,
were transfected with either a wild type (WT)
human EGF receptor (B82L-WT) or a human EGF
receptor with a Lysine to Methionine point
mutation at residue 721 (B82L-M721). This mutant
receptor is unable to signal, although it retains
the ability to bind EGF. Used in this study were
BD Transduction Laboratories mAbs
phosphotyrosine clone PY20, Caveolin clone 2297,
Phospho-Caveolin (Y14) clone 56, Stat5 clone 89,
Phospho-Stat5 (Y694) clone 47, Stat1 clone 42,
Phospho-Stat1 (Y701) clone 14. The goat
anti-mouse-HRP secondary Ab was from BD
Transduction Laboratories and the donkey
anti-mouse-Cy3 Ab was from Jackson
Immunoresearch. Immunoblotting Following
stimulation, cells were rapidly lysed in boiling
lysis buffer (1 SDS,1 mM sodium ortho-vanadate,
and 10 mM Tris-HCl pH 7.4). Twenty micrograms per
lane of whole cell lysate was size fractionated
by SDS-PAGE. Proteins were electrophoretically
transferred to ImmobilonTM-P membranes
(Millipore). Membranes were probed with the
indicated monoclonal Abs for 30 minutes at 37oC
followed by a goat-anti mouseHRP secondary Ab.
Images were visualized using SuperSignal
chemiluminescent substrate (Pierce).
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Methods - Immunofluorescence, Flow Cytometry,
BD CBA
Intracellular Analysis of Activated Cell
Signaling Molecules Through the Use of
Phospho-Specific Antibodies and Flow Cytometry
Immunofluorescence Cells were treated as
indicated and fixed with 3.75 paraformaldehyde
for 30 minutes at 370C. For analysis with
phospho-Stat1, A431 cells were fixed with 11
methanolacetone for 10 minutes at 200C.
Following fixation, cells were blocked in 1 BSA
for 30 minutes at 370C and probed with the
indicated mAbs (5 mg/ml) for 1 hour at room
temperature, followed by staining with donkey
anti-mouse-Cy3. Coverslips containing labeled
cells were rinsed in PBS, immersed in fluorescent
mounting media (Vectashield Vector
Laboratories), and mounted on glass slides with
nail polish prior to analysis. Intracellular Flow
Cytometry and CBA Following stimulation, cells
were harvested, washed in PBS, and fixed in 1
formaldehyde for 15 minutes at 40C followed by
ethanol at 200C. Cells were then permeabilized
in Perm/Wash buffer (BD Biosciences Pharmingen)
and incubated with 1 mg of the indicated
monoclonal Abs for 1 hour at room temperature.
Cells were then washed in Perm/Wash buffer and
incubated with goat anti-mouse-FITC (Jackson
Immunoresearch) for 30 minutes at room
temperature. For the CBA, denatured cell lysates
were mixed with 50 ml of Stat1-coated beads and
50 ml of Phospho-Stat1-biotin as a detector.
Beads were then washed and incubated with
streptavidin-PE. All samples were acquired on a
BD FACSCalibur and analyzed using BD Cell Quest
Pro or BD CBA software (BD Biosciences
Pharmingen).
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Figure 1 PY20 Phosphoprotein Analysis
Intracellular Analysis of Activated Cell
Signaling Molecules Through the Use of
Phospho-Specific Antibodies and Flow Cytometry
Figure 1
Figure 1 PY20 Phosphoprotein Analysis. (A)
A431 cells (lanes 1 and 2) and Human Endothelial
cells (lanes 3 and 4) were either untreated
(lanes 1 and 3) or treated with EGF (lane 2) or
sodium pervanadate (lane 4) and lysates were
subjected to WB with PY20. (B) Immunofluorescent
staining of PY20 in Human Endothelial cells
either untreated or stimulated with pervanadate
for 20 minutes. (C) Intracellular flow analysis
of tyrosine phosphorylation in A431 and Human
Endothelial cells. (D) Intracellular flow
analysis of tyrosine phosphorylation in Human
Endothelial cells in the presence or absence of a
specific phosphotyrosine competitor, phenyl
phosphate. The right panel shows competition of
the pervanadate-induced peak.
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Figure 2 Caveolin Phosphoprotein Analysis
Intracellular Analysis of Activated Cell
Signaling Molecules Through the Use of
Phospho-Specific Antibodies and Flow Cytometry
Figure 2
Figure 2 Caveolin Phosphoprotein Analysis. (A)
WB of Human Endothelial cells either untreated
(lanes 1 and 3) or treated with sodium
pervanadate (lanes 2 and 4) and probed with
Caveolin or Phospho-Caveolin (Y14), as indicated.
(B) Immunofluorescent staining of
Phospho-Caveolin (Y14) in A431 cells either
untreated or stimulated with EGF for 5 minutes.
(C) Intracellular flow analysis of Caveolin or
Phospho-Caveolin (Y14) in unstimulated or EGF
stimulated A431 cells. (D) Intracellular flow
analysis of Caveolin and Phospho-Caveolin (Y14)
in Human Endothelial cells in the absence (upper
panels) or presence (lower panels) of peptide
competitors (linear Phospho-Caveolin or AIB-1
peptides) to confirm specificity. The
phosphotyrosine competitor, phenyl phosphate did
not block the positive Ab shifts (data not shown).
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Figure 3 Stat5 Phosphoprotein Analysis
Intracellular Analysis of Activated Cell
Signaling Molecules Through the Use of
Phospho-Specific Antibodies and Flow Cytometry
Figure 3
Figure 3 Stat5 Phosphoprotein Analysis. (A) WB
of A431 cells either untreated (lanes 1 and 3) or
stimulated with EGF for 5 minutes (lanes 2 and 4)
and probed with Stat5 or Phospho-Stat5 (Y694), as
indicated. (B) Intracellular flow analysis of
Stat5 and Phospho-Stat5 (Y694) on U937 cells
either untreated or stimulated with GMCSF for 20
minutes. (C) Intracellular flow analysis of
Stat5 and Phospho-Stat5 (Y694) on Human
Endothelial cells untreated or treated with
sodium pervanadate for 20 minutes.
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Figure 4 (slide 1) Stat1 Phosphorylation
Intracellular Analysis of Activated Cell
Signaling Molecules Through the Use of
Phospho-Specific Antibodies and Flow Cytometry
Figure 4 (slide 1)
Figure 4 (slide 1) Stat1 Phosphorylation. (A) WB
of U937 cells either untreated (lanes 1 and 3) or
stimulated with IFNg (lanes 2 and 4) and probed
with Stat1 or Phospho-Stat1 (Y701), as
indicated. (B) WB of A431 cells either untreated
(lanes 1 and 3) or treated with sodium
pervanadate for 20 minutes (lanes 2 and 4) and
probed with Stat1 or Phospho-Stat1 (Y701), as
indicated. (C) Immunofluorescent staining of
Stat1 or Phospho-Stat1 (Y701) in untreated or EGF
stimulated A431 cells.
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Figure 4 (slide 2) Stat1 Phosphorylation
Intracellular Analysis of Activated Cell
Signaling Molecules Through the Use of
Phospho-Specific Antibodies and Flow Cytometry
Figure 4 (slide 2)
Figure 4 (slide 2) Stat1 Phosphorylation. (D)
Intracellular flow analysis of Stat1 and
Phospho-Stat1 (Y701) in A431 and U937 cells in
the absence or presence of pervanadate and IFNg,
respectively. (E) Intracellular flow analysis of
Phospho-Stat1 (Y701) in U937 cells either
untreated or stimulated with IFNg in the absence
or presence phosphorylated Stat1 peptide (upper
panels) or phosphorylated Caveolin peptide (lower
panels) to confirm specificity. The
phosphotyrosine competitor, phenyl phosphate, was
unable to block the positive Ab shifts (data not
shown).
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Figure 5 BD CBA Analysis of Stat1
Phosphorylation
Intracellular Analysis of Activated Cell
Signaling Molecules Through the Use of
Phospho-Specific Antibodies and Flow Cytometry
Figure 5
Figure 5 CBA Analysis of Stat1
Phosphorylation. (A) The CBA assay. Denatured
cell lysate is incubated with Ab coated beads and
fluorescent tagged detector Abs. After washing,
samples are examined by standard flow
analysis. (B) WB of B82L-WT and EGF receptor
mutant B82L-M721 cells treated with EGF for the
indicated times and probed with
Phospho-Stat1(Y701). (C) CBA analysis of B82L-WT
and EGF receptor mutant B82L-M721 cells treated
with EGF for the indicated times. A standard
curve was generated using a Jurkat pervanadate
lysate (data not shown) with the maximum reading
designated as 1000 units. Experimental samples
were assayed simultaneously and relative units
assigned to each sample based on fluorescence and
the standard curve.
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Conclusions
Intracellular Analysis of Activated Cell
Signaling Molecules Through the Use of
Phospho-Specific Antibodies and Flow Cytometry

• Conclusions
• We have developed phospho-specific mAbs that can
  be used in multiple applications, including flow
  cytometry, to effectively analyze the
  phosphorylation state of cell signaling proteins.
• Flow cytometry can be utilized with
  phospho-specific mAbs to obtain quantitative data
  for intracellular signaling proteins on a single
  cell basis in a manner that is more rapid and
  informative than Western blotting.
• The CBA system is a powerful multiplexed assay
  that allows for the rapid detection of cell
  signaling protein phosphorylation from cell
  lysates and requires a smaller sample size than
  that necessary for Western blotting.
• The ability to stain both cell surface and
  intracellular signaling molecules allows for the
  rapid, multiparameter flow cytometry- based
  analysis of distinct cell subpopulations whose
  analysis has previously been difficult due to
  detection limitations or sample size.