1. Analysis of Cellular Signaling using
Activation-State Specific Antibodies
Greg Innocenti
Cell Signaling Technology
December 19, 2006

2. Outline
What are activation state-specific antibodies?
Imaging Cytometry
• immunofluorescence & antibody validation
• antibody conjugation
• high content analysis (HCA)
Flow Cytometry
• protocols
• clinical assays

3. Outline
What are activation state-specific antibodies?
Imaging Cytometry
• immunofluorescence & antibody validation
• antibody conjugation
• high content analysis (HCA)
Flow Cytometry
• protocols
• clinical assays

4. Activation State-Specific Antibodies
phospho-p38
Total (bind regardless of activation state)
Phospho-specific (eg. phospho-EGFR)
p38
Cleavage-specific (eg. cleaved Caspase-3)
Acetylation-specific (eg. acetyl-Stat3)
+ - + -
Methylation-specific (eg. methyl-Histone H3)
Substrate-specific (eg. phospho-Akt Substrate)
Motif-specific (eg. phospho-Tyrosine)

5. Activation State-Specific Antibodies
phospho-p38
Total (bind regardless of activation state)
Phospho-specific (eg. phospho-EGFR)
p38
Cleavage-specific (eg. cleaved Caspase-3)
Acetylation-specific (eg. acetyl-Stat3)
+ - + -
Methylation-specific (eg. methyl-Histone H3)
Substrate-specific (eg. phospho-Akt Substrate)
Motif-specific (eg. phospho-Tyrosine)

6. Total Antibodies
Untreated Wnt 5’ Whole Blood
HeLa Cells
CD5
β-Catenin (C-term)
CD13
LY294002 Insulin
Side Scatter
C2C12 Cells
Akt (pan) (11E7)
CD4

7. Phospho-specific Antibodies
Untreated EGF-Treated
Phospho-EGFR (PE)
EGF Receptor (FITC)
Green = Phospho-EGF Receptor (Tyr1068)
Blue = DRAQ5

8. Caspase-3 Cleavage in Apoptosis
Untreated Staurosporine
TUNEL
Cleaved-Caspase 3
Green = Cleaved Caspase-3 (Asp175) (5A1) RmAb
Red = F-Actin (Phalloidin)
Blue = DRAQ5

9. Antibody Validation & Applications
CST’s activation-specific antibodies are purified to distinguish
between only one or two posttranslationally modified amino acid
residues
Most antibodies raised against such modified sites have a lower
affinity when compared with antibodies recognizing whole
proteins
Thus, all product development follows rigorous in-house testing on
a wide range of assay applications by our clinical applications
specialists
 Stringent validation requirements
 Protocol optimization
 Cross platform functionality

10. Outline
What are activation state-specific antibodies?
Imaging Cytometry
• immunofluorescence & antibody validation
• antibody conjugation
• high content analysis (HCA)
Flow Cytometry
• protocols
• clinical assays

11. Antibody Validation for Immunofluorescence/HCA
Part I - Titration to determine Part II - Specificity Testing
optimal dilution/concentration • treat cells with specific
ligands, drugs, inhibitors, etc.
• test antibody on cells that do
and do not express target
• verify expression or treatment
efficacy with another antibody
(same target, or different
target in same pathway)
Confocal Imaging on Chamber Slides
Titration Curve
45000.00 6.00
40000.00
5.00
35000.00
30000.00 4.00
2966
Mean Channel Fluorescence
Signal to Noise Ratio
25000.00
3.00 Control
20000.00
S/N Ratio
15000.00 2.00
10000.00
1.00
5000.00
0.00 0.00
0 5 10 15 20 25 30 35 40 45
Antibody Dilution (ug/ml)

12. Antibody Validation for Immunofluorescence/HCA
[Ab]
Antibody
Isotype
Antibody
1:25
1:50
1:100
1:200
1:400
1:800
16000.00 4.50
14000.00 4.00
12000.00 3.50
3.00
10000.00
Isotype
4694
Mean Channel Fluorescence
2.50
Signal to Noise Ratio
8000.00 Control
2.00
6000.00 S/N Ratio
1.50
4000.00
1.00
2000.00 0.50
0.00 0.00
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Antibody Dilution (ug/ml)
MEK1/2 (red)
actin (green)
DNA (blue)
HeLa cells

13. EGF Receptor Inhibition
EGFR Mutant NSCLC Cell Lines
Untreated Iressa
HCC827 cells
Del 746-750, amp.
H1975 cells
L858R + T790M
phospho-Tyrosine (red, Alexa647 conjugate)
phospho-S6 (green, Alexa488 conjugate)

14. GPCR Activation
MEK PI3K
inhibitor inhibitor
LPA treatment 0 min 2 min 5 min 15 min 15 min 15 min
merge
phospho-Erk
phospho-Akt
DNA
C6 cells

15. Neuroscience - Development
Postnatal Day 1 Postnatal Day 14
Postnatal Rat Brain
p-Histone H3
Red=GFAP
Blue=Doublecortin
P1 P4 P14

16. Cell Cycle / Checkpoint
Untreated Untreated
UV Normal Colon Dexamethasone Colon Carcinoma
Metaphase
UV UV+PPT
Anaphase
#9309 Rb (4H1)
#3068 p-Aurora #2558 p-Kip2 (T310) p-Rb (S807/811) p-p53 (Ser37) p-Rad17 (S645)
#9309 Rb (4H1)
#9719 p-H2A.X (S139) Aurora A/AIK
A/B/C #9308
#4718 #9289 #3421
P Chk1/2 p53 Aurora
Cdc25A Kip1 P
cdc25B P
ATM
P
CDK4/6 CDK2 cdc2
CyclinD CyclinE CyclinB
G1 Phase R S Phase G2 Phase M Phase
P
P P Rad17
Abl
HDAC P
Rb
P X P P
Rb cdc2 H2A H3
E2F1 E2F1
OFF DP1 ON
DP1 DNA Damage

17. CST Conjugated Antibodies
Conjugated antibodies helpful for multiplex analyses
CST conjugates are optimized for cytometric applications
• high-quality pre-validated antibodies
• bright photostable Alexa dyes
• F/P trials to ensure bright signal
• antibody titration
• stability tested (accelerated and real-time)
• screened with flow cytometry, HCA, and IF
• lot-to-lot stability

18. Survivin (Alexa488-conjugate)
FAS, TNFa
• inhibits apoptosis and regulates mitosis
• over-expressed in most human cancers
FADD
• expression correlates with both accelerated Caspase-8/10
ER Stress Mitochondria
relapse and chemotherapy resistance
[Ca++] Smac/
P1 Rat Brain Diablo Cyto C
Caspase-12 Survivin
Caspase-9
Caspase-3
Caspase-6 Caspase-7
Lamin A a-Fodrin DFF PARP
CST’s conjugated Survivin antibody is currently being used to screen patient samples

19. The Bigger Picture
Single well immunofluorescence
+ ability to examine subcellular (co)localization in 4-dimension
(XYZt)
- difficult to quantify without specialized software
- imaging is time consuming and data files become massive
High Content Analysis
+ some systems able to analyze localization
+ rapid scanning (comparatively), sensitive, and quantifiable
+ ability to multiplex and dissect various pathways in tandem

20. High Content Analysis
• automated plate-based image analysis
• quantifiable signal intensity and subcellular
1:800
1:100
1:400
1:200
1:50
1:25
localization Untreated
• more predictive of drug activity in a cellular PDGF
p-Erk
environment compared to ELISAs U0126+PDGF
LY/Wort+PDGF
• can be used to determine: Untreated
efficacy and therapeutic dose
PDGF
p-Akt
U0126+PDGF
cell-permeability of drugs LY/Wort+PDGF
potential toxicity (DNA damage, apoptosis,
micronuclei)
downstream effects of drug/target interaction
off-target effects

21. Value of CST Antibodies in HCS
Current platforms have increased colors and resolution in an attempt to
quantify complex events
Example: nuclear translocation of Erk or NFkB
Requirements
• nuclear marker
• total antibody
• high resolution optics
• complex software
• lots of data storage
Using a phospho antibody will eliminate these costly requirements and
speed up the screen (on/off as opposed to determination of localization)
KEY: reliable simple affordable assay with clear robust results

22. Multiple, Parallel Analyses by Cellular Imaging (ICW)
Gleevec/PDGF
U0126/PDGF
LY294002
Untreated
Gleevec
PDGF
PDGF
PMA
blank
p-PDGFR
p-Erk
p-Akt
RTK signaling analysis using LI-COR Odyssey
Untreated Anisomycin UV
p-p38
p-Jnk
p-ATF2
p-H2A.X
DNA damage profiling analysis using LI-COR Odyssey

23. Future Plans: Broad Signal Profiling by HCA
• large antibody panel for profiling screens
• can be used on any cytometric platform
• detection = fluorescent, chromogenic, chemiluminescence
Starved
• up to 96 antibodies per plate
total and phosphorylation-specific antibodies
MAPK, Akt, NFkB, Jak/Stat, etc.
receptor tyrosine kinases (EGFR, VEGFR, FGFR, IGF-IR, cKit)
adaptor proteins and downstream targets EGF
transcription factors
motif antibodies (general serine or tyrosine phosphorylation)
• can be customized for analysis of different biological processes
toxicity
cell cycle/arrest Anisomycin
apoptosis
cell adhesion
96 prediluted cells treated with
CST antibodies compound X
UV

24. Antibody Development: FoxO1 Screening
PI3K inhibitor IGF-1 + serum
(Akt off) (AKT on)
How can we generate antibodies that
work well for cytometric applications?
Screen using cytometric applications
HTS & HCS Platforms
Nuclear Cytoplasmic
nuclear trigger
(half-width intensity)

25. Outline
What are activation state-specific antibodies?
Imaging Cytometry
• immunofluorescence & antibody validation
• antibody conjugation
• high content analysis (HCA)
Flow Cytometry
• protocols
• clinical assays

26. Optimization: Fixation/Permeabilization
CST 2-4% formaldehyde/90% methanol
Fix&Perm Kits 0.25-4% formaldehyde/detergent (triton or saponin)
Krutzik and Nolan (2004) Cytometry 55A:61-70.

27. Fixation/Permeabilization
p-p38
p-Erk
2% Formaldehyde 4% Formaldehyde Commercial Fix&Perm Kit
90% Methanol 0.3% Triton X-100 (aldehyde/saponin)

28. Surface and Signaling Markers
• Methanol diminishes or abolishes signal from some key surface markers
• Many signaling event are very transient so immediate fixation is critical, no
time to prelabel with surface markers, lyse RBCs, or perform FiColl
separations
• Staggered protocol: fix cells with aldehyde to stop all enzymes and preserve
phosphoepitopes, label with surface markers, permeabilize with methanol,
and then label with intracellular signaling antibodies (destroys some
fluorochromes)
• Need a better solution…

29. New Fix & Perm Protocol
Untreated
PMA
4% Formaldehyde + 0.1% Triton X-100 + 50% Methanol

30. Protocol Comparison
4%PFA at RT for 10m
3% PFA at 37ºC for 10m 0.1% Triton X-100 at RT for 30m
90% ice cold MeOH at -20ºC for 10m 50% ice cold MeOH at 4ºC for 10m
CD19 (FITC) CD19 (FITC)

31. Flow Cytometry: Clinical Applications
• Staining of intracellular signaling molecules can be easily
incorporated with traditional cell surface marker labeling
4% Formaldehyde for 10m
0.1 Trinton X-100 ft RT for 30m
50% ice cold MeOH at 4oC for 10m
• Important implications in the Clinic
• Flow Cytometry and Disease-specific Signaling
• Chronic myelogenous leukemia (CML)
• Chronic lymphocytic leukemia (CLL)
• Acute myelogenous leukemia (AML)

32. Bcr/Abl Pathway Profiling by Flow Cytometry
CML cells (K562) + Gleevec
phospho-
Bcr
phospho-
Stat5
cleaved-
Casp3
0 hr 1 hr 24 hr 48 hr

33. Large Scale Bcr/Abl Pathway Profiling
K562 cells +/- Gleevec
bar height represents amount of Gleevec inhibition

34. Biomarkers for CLL
*
*
Staudt’s lab first identified Zap-70 as a potential biomarker of the aggressive form of CLL
CLL patients with Zap-70 (+) B-cells have poorer prognosis
Crespo et al. (2003) N Engl J Med 348:1746-75

35. CLL Assay using Zap-70 (136F12) RmAb
In House: In the Clinic:
B cells (Ramos)
T cells (Jurkat)
Primary CLL Cells: Source: Esoterix Center for Innovation
Difficulties persist in Zap-70 analysis
- Zap-70 levels decline significantly after blood is drawn
- Inconclusive results with weak expression
Zap-70 (low) Zap-70 (high) - Additional biomarkers to make assay more reliable?
(slow growing) (fast growing)

36. Alternative CLL Biomarkers
6.00
5.00
Mean Fluorescence (normalized)
4.00
CLL23LB4 (Zap70-)
3.00
MO1043 (Zap70+)
2.00
1.00
0.00
p-PLCg p-FGFR PAK Zap70 p-Stat1 p-Stat3 p-NFkB p65 p-IGF-IR
(Y783) (y653/654) (S727) (S727) (S536) (Y1131)
CLL23LB4 (Zap70-)
MO1043 (Zap70+)
Rosenwald et al. (2001) J Exp Med. 194(11):1639-1647.

37. Flt3 Signaling
Flt3 • Mutation or overexpression of Flt3 kinase
is observed in AML, (~70%), B-ALL, T-
ALL, and some blast phase CML
• Most common mutations are internal
tandem duplications (ITD) and active loop
mutations (AL), both of which result in
constitutive activation
• Phospho-specific antibodies can be used
to profile downstream signaling and to
monitor the efficacy of specific Flt3
inhibitors
Scheijen and Griffin (2002) Oncogene 21:3314-33

38. Flt3 Inhibitor Pharmacodynamic Assay Design
Phospho-Flt3
Flt3 ITD Overexpressed Flt3 Alexa488 Conjugate
(MOLM-14(MOLM14)
Dose Response Cells) (SEM Cells)
Dose Response (SEM) (SEM cells)
1.4 1.4
Normalized mean fluorescence
Normalized mean fluorescence
1.2 1.2
1 1 p-S6
p-S6
p-Flt3 p-Flt3
0.8 0.8
p-Stat5 p-Stat5
0.6 p-Stat3 p-Stat3
0.6
PY p-Erk
0.4 p-AKT PY
0.4
p-AKT uninhibited Flt3 inhibitor
0.2
0.2
0
0
0 0.01 0.1 0.5 2
0 0.01 0.1 0.5 2
Concentration of Flt3 inhibitor (µM) Concentration of Flt3 inhibitor (µM)
Conclusion:
Phospho-S6 is a key indicator
of Flt3 inhibition, regardless of
mutation
This antibody is a reliable
Flt3 inhibitor decreases
downstream signaling,
Percentage of Cleaved Caspase-3 positive cells
robust biomarker for receptor
causes cell cycle
80
70 tyrosine kinase (RTK) activity
60
arrest, and induces
Percent (%)
50
apoptotic cell death 40
30
20
10
0
0 2 3 5 22
Time (hours)

39. Conclusion
• Activation state-specific antibodies are very useful in multiplex
_phosphoproteomics analyses on various fluorescence based platforms
• stringent validation assures highest quality antibody and lot-to-lot
reproducibility
• determine prognostic significance of signaling anomalies
• monitor patients for resistance or recurrence
• study effectiveness and optimal dosage of specific kinase inhibitors
• allow tumor “typing” to determine best treatment option
• improve clinical trial and basic research design (outcome and economics)
Gregory Innocenti
Clinical Applications/Image Cytometry
ginnocenti@cellsignal.com
978-867-2475

40. Thank You