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