the afcs antibody lab rod ceja blythe king eduardo arteaga
TRANSCRIPT
The AfCS Antibody Lab
Rod Ceja Blythe King
EduardoArteaga
AfCS Antibody Lab: Year 4 Goals
Transition to RAW 264.7 macrophage cell line
Double the number of phosphoproteins monitored
Initiate ligand screen for phosphoproteins
Collaborate with Bio-Rad on Luminex assays of site specific phosphorylation (Bio-Plex)
Initiate double ligand screen for cytokine release
Site Specific Protein Phosphorylation Monitored by the AfCS Antibody Lab
Quantify ligand-induced changes in site specific phosphorylation of selected proteins.
Aims: sample diversity of cellular response to ligands and identify interactions between ligands
Approach: Multiplex Western blotting with mixtures of site specific, phosphosensitive antibodies
Ila Oxendine, Frank Amador, and Jeff Scalesmanage to smile in the cold room
Dual Ligand Screen
characterization
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2003 AfCS Annual Meeting
40 Chromo-somes
Frozen stocks
PP cAMP Ca2+ receptor class
1 2MA 2-Methylthioadenosine 5'-triphosphate GPCR2 848 R-848 TLR3 C5A Complement 5a GPCR4 GMF Granulocyte macrophage colony stimulating factor cytokine5 I1B Interleukin 1 beta cytokine6 I04 Interleukin 4 cytokine7 I06 Interleukin 6 cytokine8 I10 Interleukin 10 cytokine9 IFA Interferon alpha cytokine
10 IFB Interferon beta cytokine11 IFG Interferon gamma cytokine12 ISO Isoproterenol GPCR13 Ig2 Fc gamma receptor 1 multisubunit14 LPA Lysophosphatidic Acid GPCR15 LPS Lipopolysaccharides TLR16 MC1 MCP117 MCF Macrophage colony stimulating factor RYK18 M1A Macrophage Inflammatory Protein alpha19 P2C PAM2CSK4 TLR20 P3C PAM3CSK4 TLR21 PAF Platelet Activating Factor GPCR22 PGE Prostaglandin E2 GPCR23 S1P Sphingosine 1-phosphate GPCR24 TGF Transforming growth factor beta RS/TK25 UDP Uridine 5'-diphosphate GPCR
Ligands
P-proteins cAMP calcium receptor class
1 2MA 2-Methylthioadenosine 5'-triphosphate GPCR2 848 R-848 TLR3 BRA Bradykinin GPCR4 C5A Complement 5a GPCR5 CER Ceramide6 CPG CpG GPCR7 CGS 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido adenosine GPCR8 DIM Dimaprit GPCR9 DOP Dopamine GPCR
10 ELC EBI1 ligand chemokine GPCR11 EST Estradiol GPCR12 ET1 Endothelin 1 GPCR13 FML fMLP cytokine14 GMF Granulocyte macrophage colony stimulating factor cytokine15 HIS Histamine GPCR16 I1B Interleukin 1 beta cytokine17 I02 Interleukin 2 cytokine18 I03 Interleukin 3 cytokine19 I04 Interleukin 4 cytokine20 I06 Interleukin 6 cytokine21 I10 Interleukin 10 cytokine22 I13 Interleukin 13 cytokine23 IFA Interferon alpha cytokine24 IFB Interferon beta cytokine25 IFG Interferon gamma cytokine26 IGF Insulin-like Growth Factor27 ISO Isoproterenol GPCR28 Ig2 Fc gamma receptor 1 multisubunit29 LB4 Leukotriene B 4 GPCR30 LPA Lysophosphatidic Acid GPCR31 LPC Lysophosphatidylcholine GPCR32 LPS Lipopolysaccharides TLR33 LTX Lymphotoxin beta34 MC1 MCP135 MCF Macrophage colony stimulating factor RYK36 MIF Macrophage Inhibitory Factor37 M1A Macrophage Inflammatory Protein alpha38 M1B Macrophage Inflammatory Protein beta39 MSHA Melanocyte Stimulating Hormone40 NOR Norepinephrine41 P2C PAM2CSK4 TLR42 P3C PAM3CSK4 TLR43 PACAP PACAP44 PAF Platelet Activating Factor GPCR45 PGE Prostaglandin e2 GPCR46 POLY:IC Poly I:C47 PPC Palmitoyl-lyso PC48 RNL RANK Ligand49 S1P Sphingosine 1-phosphate GPCR50 SCF Stem cell factor 151 SDF Stromal cell derived factor 152 TGF Transforming growth factor beta RS/TK53 TNF Tumor Necrosis Factor54 UTP/UDP Uridine 5'-triphosphate GPCR55 VEG Vascular Endothelial Growth Factor56 VIP Vasoactive Intestinal Peptide
Ligands
InitialLigand List/Response Summary Final
Ligand List cAM
P
Ca2+
p9
0R
SK
Erk
(1,2
)
Rib
S6
Akt
GS
K3
(a,b
)
Sta
t 1(a
,b)
Sta
t 3
Sta
t5
PK
Cm
PK
C (d ,
q )
NF
kB
p3
8M
AP
K
JNK
(L,S
)
Sm
ad
2
Mo
esi
n
Ezr
in
p4
0P
ho
x
ISO L+?PGE E ? E+?C5A E+LPA E+ E+?2MA E+ E+ E+ E+ E+? E+ NC? E+?PAF E+ E+ E+ E+ E+? E+ E+UDP E+ E+? E+UTP E+ E+ E+S1P NC? E+ E+ E+MCF E+TGF L+GMF E++I04I06 E+ E+++I10 E+++IFA E+ E+IFB E+ E+IFG E+++ E+++ E+++I1BLPS L+ L+ L+ L+? L+ L+? L+ L+ L++ L++P2C L++ L+ L+ L+ L+ L+ L++ L++++P3C L++ L+ L+ L+ L+ L+ L++ L++++848 L+ L+ L+ L+? L+? L+? L+ L++ L++++NL
GC
SF
GM
CS
F
IFN
g
IL1a
IL1b
IL2
IL3
IL4
IL5
IL6
IL1
0
IL1
2(p
40
)
IL1
2(p
70
)
IL1
7
KC
MIP
1a
RA
NT
ES
TN
Fa
? ?
?
?
? ? ?
Ligand Screen Results
RAW 264.7
RAW Cell Ligand ScreenRobert Hsueh’s Time Line
Robert HsuehCell Lab
Heping HanAntibody Lab
Testing antibodies and ligands
Vary ligand conc.
Time courses
Ab Lab:11 phosphoproteins: 80 samples/wk
21 phosphoproteins: 170 samples/wk
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AfCS Antibody Database: New Web-based VersionBecky Fulin, Lonnie Sorrells, Robert Sinkovits*, Ruth Levitz, and Heping Han
The Alliance for Cellular Signaling: The Antibody Laboratory (University of Texas Southwestern Medical Center at Dallas)
and the Bioinformatics and Data Coordination Laboratory (University of California at San Diego)*
Results of Antibody Testing and Scoring Are Recorded in the Antibody Database
The AfCS Antibody Database is a resource for the research community to learn how well commercially available antibodies performed in our lab
Becky Fulin presents poster #20Tues. AM: Heping Han will give brief presentation on database
p90RSK
**
*
*
*
* **
*
*dqm
*
RiboS6p40
phox*
* Phosphospecific Antibody Targets
*RiboS6
*
Toll-like Receptor Signaling
**
** Phosphospecific Antibody Targets
Targets of Current Phosphospecific Antibody Mixtures
for Multi-Plex Western Blotting
Mix 1 p90 RSK (S380) ERKs (T202/Y204) Ribosomal S6
(S235/236) Akt (S473)
Mix 2 PKCm(S916) STAT 3 (Y705) STAT 1 a/β (Y701) NFB p65 (S536) JNKs (T183/Y185) p38 MAPK (T180/Y182)
Mix 3 STAT 5 (Y694) Ezrin/radixin/moesin
(T567/T564/T558) GSK 3 a/β (S21/9)
Mix 4 PKC d(S643/676) Smad 2 (S465/467) p40 Phox (T154)
Total: 16 Antibodies for 21 phosphoproteins(counting resolvable isoforms)
Export to Bob Sinkovits of the Bioinformatics Groupin San Diego for graphing & display
Image analysis / quantification
Multiplex western blot
How Phosphoprotein Data Are Processedfor the Ligand Screen
Macrophage Ligand Screen: PhosphoproteinsNicholas Wong, Robert Hsueh*, Robert Sinkovits‡, and Heping
HanThe Alliance for Cellular Signaling: The Antibody Laboratory (University of Texas Medical Center at Dallas),
the Cell Preparation and Analysis Laboratory (University of Texas Medical Center at Dallas)*, and the Bioinformatics and Data Coordination Laboratory (University of California at San Diego)‡
Ila Oxendine
Nick Wong
Becky Fulin
Jeff Scales
RAW 264.7 Cells: Statistically Significant Phosphoprotein Responses to Single Ligands
Ligand Rib S6 p38MAPK JNK L,S STAT 3 STAT 5
2MA
ISO
PGE
C5A
LPA
PAF
UDP
S1P b a
MCF
TGF
GMF
I04
I06
I10 ?
IFA
IFB
IFG
I1B
MC1 ?
LPS
P2C
P3C
848
Cytokine Receptors
Receptor Kinases
G Protein Coupled
Receptors
?
NFkB
a
?
a
GSK3 abAktp90RSK
1
?
2
Toll Like Receptors
ERK1/2
bb
STAT1ab
?
PKCm PKCdq p40Phox Smad2 Moesin Ezrin
?
?
? ?
?
?
Increased phosphorylation at any time point (1, 3, 10, or 30 min)Decreased phosphorylation
Decreased followed by increased phosphorylation
p < 0.05
Madhu Natarajan
PAM3CSK4, a Synthetic TLR Ligand, Inhibits Interferon b-stimulated Phosphorylation of STAT3 but not STAT1
Red = Interferon b alone, stimulates phosphorylation of STATs 1 & 3Blue = combination of two ligands Green = P3C alone (not seen if STAT band invisible, i.e. STAT3)
P3C
IFB
P3C+IFB
PAM3CSK4 binds to heterodimer of TLR 1 & 2
Lipopolysaccharide Inhibits Interferon b-stimulated Phosphorylation of STATs 1, 3, & 5
Red line = Interferon b alone, stimulates phosphorylation of all three STATsBlue line = always combination of two ligands Green line = LPS alone, not seen if STAT band invisible
LPS is agonist of TLR 4
Summary of Negative Interaction between Interferons and TLR Ligands
Ligand Receptor Inhibition of STATPhosphorylation
LPS TLR4 STAT 1, 3, & 5
PAM2CSK4 (P2C) TLR 2/6 STAT 1 & 3, not 5
PAM3CSK4 (P3C) TLR 2/1 STAT 3, not 1 or 5
R848 TLR 7 STAT 1 & 3
Inhibition of Interferon a- or b-stimulated Phosphorylation of STATs
Comparison of Results between the Various Ligand Screen Assays
Phosphorylation of STATs TLR ligands alone have no effect on phosphorylation of STATs TLR ligands selectively inhibit interferon-stimulated phosphorylation
of STATs Cytokines
Interferona or balone has no effect on secretion of cytokines Interferon enhances TLR-stimulated release of cytokines (next slide)
Ligand Pair P-STATs Cytokines
cAMP Calcium
TLR ligand + IF aorb
Negativeinteraction
Positiveinteraction
No responsesingle ligands
No responseSingle ligands
Interferon a or b Enhances TLR ligand-induced Secretion of IL-6, IL-10, and RANTES
Example: Interferon b enhances LPS-stimulated secretion of IL-4, IL-10, and RANTES
Poster # 24Macrophage Ligand Screen: Cytokines
Ruth Levitz, Robert Hsueh‡, Lonnie Sorrells, Robert Sinkovitz*,Heping Han and Ron TaussigThe Alliance for Cellular Signaling: The Antibody and the Cell Preparation and Analysis ‡ Laboratories (University of Texas Southwestern Medical Center at Dallas)
and the Bioinformatics and Data Coordination Laboratory* (University of California at San Diego)
Ruth Levitz
Becky Fulin and Frank Amador
Monitoring Phosphoproteins in the Antibody Lab: the Future
Ligand Screen: complete three replicates of each ligand pair
FXM: assay responses in knockdowns, other perturbations
Collaborate with Bio-Rad and Cell Signaling Technology
development of Luminex technology for monitoring site-specific protein phosphorylation = Bio-Plex
Potential to assay more phosphoproteins simultaneously with less total protein
See posters 14 and 15, Marc Mumby’s presentation on Weds.
Monitoring Site Specific Protein Phosphorylation:
Comparison of Western Blotting and Bio-Plex Heping Han, Biren Zhao, Claudia Suen* and Judith Zhu-Shimoni*
Antibody Laboratory of the Alliance for Cellular Signaling and Bio-Rad Laboratories*
Heping Han & Biren Zhaoon Halloween