Antibody LabB Cell Ligand Screen

Quantify ligand-induced changes in site-specific phosphorylation of selected signaling proteins

Erica Turon, technician and Heping Han, Lead Scientist

Antibody LabB Cell Ligand Screen

Aim: sample diversity of responses to inputs Approach: Probe Western blots with mixture of site specific, phosphorylation sensitive antibodies Goal: resolve and quantify 5-7 phosphoproteins per lane of gelProgress: 2 mixtures of antibodies for 11 phosphoproteins

Targets of Current Mixes of Phosphospecific Antibodies

Mix 1 Stat 6: Y641

p90RSK: S381

Akt: S473

ERK 1 T202/Y204

ERK 2 T183/Y185

Mix 4 PKCS916 Stat 3 Y705

NFB p65 S536

JNK long T183/Y185

JNK short T183/Y185

p38 MAPK T180/Y182

Criteria: single band of appropriate size, ligand sensitiveTested 64 different antibody preparations (1 out of 7)All antibodies from Cell Signaling Technology, except one

B Cell ReceptorMap

*

*

Untr

eate

d

Anti

-IgM

IL-4

Pos.

Cont.

A

Anti-IgM = 0.3 M, IL-4 = 0.4 nM, Positive control A = Anti-IgM + IL-4 All treatment was 5 min

Phosphospecific Antibody Mix 1

p90RSK

ERKs

Stat6

Akt

Anti-IgM (0.3M )

IL-4 (0.34 nM)

Anti-CD40 (65 nM)

2.5’ 5’ 15’ 30’

2.5’ 5’ 15’ 30’

2.5’ 5’ 15’ 30’

+ C

ontr

ol

+ C

ontr

ol

Unt

reat

ed

Unt

reat

ed

<p90RSK

<Akt

>ERKs

<Stat6

<G

Ligand Screen Time Courses

EWC020314B_EACell ID = EWC020319DAnti-IgM = 0.3 M; IL-4 = 0.34 nM ; CD-40 = 65 nM

Imaging

No film or radioactivityECL Plus (has fluorescent by-product)Imager detects fluorescenceImageQuant software to save image

Biren Zhao with Storm 860 Imager from Molecular Dynamics

Quantification

Nick Wong: searched for better software, implementedImageMaster: Automatically finds lanes, semi-automatic for bands

Interleukin 4 Phosphorylation of Stat

6

50 fold increase in phosphorylation of Stat6Single scale: Cannot see changes in phosphorylation of other proteins

0

20

40

60

80

100

120

0 2.5 5 15 30

time (min)

fold

ch

ang

e

P-Stat6

P-p90RSK

P-Akt

P-ERK1

P-ERK2

Interleukin 4

Two y axes scaled differentlyReveals phosphorylation of Akt (fuscia)

Additional Ligands:Gi-mediated Responses

Sphingosine 1-phosphate and Chemokines: SDF, BLC, ELC, SLC Increased phosphorylation of Akt, ERKs and p90RSK with

max. response at first time point More rapid initial decline of ERKs

Time course pattern similar to B cell receptorGq, Gs pathways: no effect of terbutaline, PGE2, LPA, 2-MA

Antibody Mix 1Additional Ligands with No Response

IL-10InterferonIGF-1TNF Leukotriene B4fMLPNeurokinin B

Responses with Antibody Mix 4?

Positive Control B = 0.34 nM IL-10 and 65 nM anti-CD40 treated B cells for 5 min.

EA239_April 1002

Anti-CD40 + IL-10

0’ 5’ 15’

Pos

. Con

t.

B

Phosphospecific Antibody Mix 4

p38MAPK

NFB p65

PKC

Stat3

JNK

CD40Signaling

Map

* *

*

Phosphoprotein Antibody Mix 4Responses to Anti-CD40

Early response: phosphorylation of NFB p65JNKs:

delayed response (similar to ERKs and Akt)

separate y axis

Anti-CD40

0

2

4

6

8

10

0 5 10 15 20 25 30Time (min)

Fo

ld c

han

ge

0

5

10

15

20

25

Fo

ld C

han

ge

for

JNK

P-PKCmu P-Stat3P-NF-kB P-p38P-JNK long P-JNK short

Phosphoprotein Antibody Mix 4Responses to IL-4 and Anti-IgM

IL-4: Rate of Stat 3 phosphorylation slower than Stat 6

Anti-IgM: modest responses

Anti-IgM

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 5 10 15 20 25 30

Time (min)

P-PKCmuP-Stat3P-NF-kBP-JNK longP-JNK shortP-p38

IL-4

0

5

10

15

20

25

30

35

40

0 5 10 15 20 25 30

Time (min)

Fold

cha

nge

P-PKCmuP-Stat3P-NF-kBP-JNK longP-JNK shortP-p38

Phosphoprotein Antibody Mix 4Ligand Screen

Anti-IgM, Anti-CD40 and IL-4 quantifiedn=2 for data shownData on website soon

Becky Fulin, Technician

Our Group

Antibody Lab:Lead scientist: Heping HanTechnicians: Eduardo Arteaga, Becky Fulin, Nick Wong,

Biren ZhaoDallas Labs:

Data Manager: Lonnie Sorrells Administrator: Kim Edwards

Future Goals

Ligand Screens B cell WEHI 231 Myocytes Multi-ligand Luminex fluorescent bead technology

Determine concentration of signaling proteinsFocus on PIP3 Modules in WEHI 231 cells Characterize/utilize phosphospecific

antibodies Collaboration with Cell Signaling Technology

Eduardo Arteaga