Ligand Rib S6p38

MAPKJNK (L,S)

STAT 3 STAT 5 PKC PKC

p40 Phox

Smad2 Moesin Ezrin

2MA up up up up up up up

ISO up up up down up down down

PGE up up down down

C5A up down down up down down down

LPA up up up down up up up

PAF up up up up up? down down

UDP up up up up up down down

S1P up up down up up up up

MCF down up up up down down down down

TGF down up? up

GMF up up down up down down

I04 up down down down

I06 up up up up up? down? down

I10 up? up up? down down

IFA up up down down up down down

IFB down up up down down up down down

IFG up up up down up down down

I1B up down up down down

MC1 up? up up down down

LPS up up up up up

P2C down up down up up up up down up down up down up up down up up? down down

P3C up up up down up down up up down up down down

848 down up up up up down up down up up down up up down down

up = reproducible increase

down = reproducible decrease

up? = may be increase

down? = may be decrease

up?

down

down

up

down

down

down

down

down

down

up

NFkB

down

down

down

down

down

down

down

down

down

down?

down

up

up

up

up

GSK3 (

up

up

up

up

up

up

up

down

down

down

down

down

up

up

up

up

up

up

Akt

up

down

down

up

up

up

p90RSK

down

up

down

up

down

up

down

up

up

up

up

up

up

up

up

down 1

down

down?

up

down

down

up

up

up

up 2

Toll Like Receptors

ERK (1, 2)

up

up

down

down

up

up

down

down?

STAT1 (

up

up?

up

up

up

up

Receptor Kinases

G Protein Coupled

Receptors

Cytokine Receptors

up

up

up

RAW 264.7 two-ligand screen Strategy to Monitor Protein Phosphorylation for the Macrophage Ligand Screen

Cell Preparation and Analysis Lab: Expose RAW 264.7 cells to single and paired combinations of ligands

– Goal: ~23 ligands and their ~253 paired combinations: conduct each 3 times on different days

– Experimental design:• Expose cells to 4 single ligands and their 6 possible

combinations• 4 time points: 1’, 3’, 10’, 30’ • Extract cells with SDS-PAGE sample buffer (supplemented with

inhibitors of proteases and phosphatases) including 3 cultures of untreated cells = 43 samples per experiment

Antibody Lab: Assay 21 phosphoproteins by multiplex Western blotting – Centrifuge cell lysates to remove viscous DNA– Produce blots with quadruplicate pairs of Bio-Rad Criterion gel (26-

well, 4-20% gradient)– Process each pair of blots with one of 4 mixtures of site-specific,

phosphosensitive antibodies– Create images of blots with Storm 860 imager– Quantify signal from 21 phosphoproteins with aid of ImageMaster

1D Elite software– Transmit data to Bioinformatics Lab

Bioinformatics Lab: Process data– Create time course graphs from phosphoprotein data– Display graphs on web: www.signaling-gateway.org P2C + MCF: Thumbnail Graphs (9 of 21 shown)

Click

Each thumbnail links to a page displaying all independent experiments for these conditions

For example, click on Akt to see its 4 replicates

Replicate Time Courses of Akt Phosphorylationin Response to MCF + P2C

Phosphorylation of Akt reveals synergy of MCF and P2C in 3 of 4 independent experiments

Export to Bioinformatics Groupin San Diego for graphing & display

How Ligand Screen Data Are Processed

Image analysis / quantification

Multiplex western blot

Single Ligand: P2C Thumbnail Display

• Time courses of 21 phosphoproteins are displayed for representative experiment

• Click on one time course, such as p38 MAPK, and you will be taken to display of replicate experiments for this phosphoprotein

Macrophage Ligand Screen: PhosphoproteinsNicholas Wong, Robert Hsueh*, Robert Sinkovits‡, and Heping Han

The 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)‡

DATACENTER

The AfCS Antibody Laboratory: Francisco Amador, Eduardo Arteaga, Rodrigo Ceja, Becky Fulin, Blythe King, Ila Oxendine, Jeff Scales, and Susanne Mumby (Director);

The AfCS Cell Preparation and Analysis Laboratory: Julie Collins, Richard Davis, Katherine Hawes, Jason Polasek, Amy Pope, Meghdad Rahdar, Melissa Stalder, Audra Wendt, and Paul Sternweis (Director);

Data Manager for the AfCS Laboratories at UT Southwestern: Lonnie Sorrells

Associate Director of the AfCS: Ron Taussig

Acknowledgements

AbstractA major focus of the AfCS Antibody Laboratory is to quantify ligand-induced changes in site-specific phosphorylation of chosen signaling proteins. This is one approach to defining interactions between pathways, which will contribute to the development of a model for cell signaling. The AfCS Cell Preparation and Analysis Laboratory produces extracts of RAW.264.7 cells that have been exposed to 23 receptor ligands, both alone and in paired combinations. The Antibody Lab assays protein phosphorylation in these samples by multiplex Western blotting with four mixtures of site-specific, phosphosensitive antibodies. The Antibody Laboratory quantifies the signal for 21 phosphoproteins and transmits the data to the Bioinformatics Laboratory, which in turn produces graphs of the time courses (of fold change in phosphorylation) and displays them on the web. There are essentially two ways to view the phosphoprotein data on the AfCS website (www.signaling-gateway.org, >> Data Center). Accessing the ‘RAW 264.7 ligand screen’ page retrieves a summary table listing the ligands tested and links to the data from each ligand acting alone. The ‘RAW 264.7 two-ligand screen’ page displays a 2-dimensional matrix with links to time courses of phosphoprotein responses to each paired combination of ligands viewed for all 21 phosphoproteins from a single representative experiment (as “thumbnail” sketches), which in turn is linked to a page that shows results from all replicate experiments that have been conducted with a chosen ligand pair.

Single ligand time courses culled from double ligand screenexperiments

Replicates of p38 Phosphorylation in Response to P2C (4 of 7 time courses shown

here)

Summary of Phosphoprotein Responses to Single Ligands

All mixtures of phosphospecific antibodies include a conventional antibody against Rho GDI (not listed). We use the signal for Rho GDI (a 28 kDa protein not anticipated to change significantly during 30 min assays) to normalize for lane-to-lane variability in total protein load.

RAW 264.7 ligand screen 

click

Example: click on MCF + P2C

(conducted 4 times)

Double Ligand Screen Matrixfrom AfCS Data Center: each D links to

data for corresponding two-ligandinteraction

LigandsERK(1, 2)

p90 RSK

Rib S6p38

MAPKJNK(L, S)

AktGSK3 (α, β)

NFkBSTAT1 (α, β)

STAT3 STAT5 PKCµPKC(δ, θ)

p40 Phox

Smad2

Moesin Ezrin

2MA up up up up up up up upISO up up up up up up up? up? down downPGE down down up up down down downC5A up up up down down down down down down downLPA up up downPAF up up up up up upUDP up up up up up up up UTP up upS1P up up up up up up up up upMCF up down up up up down down down downTGF upGMF upI04

I06 up upI10 upIFA up up upIFB up up upIFG up up upI1B up up

MC1 downLPS up up up up up up up up up up upP2C up up up up up up up up upP3C up up up up up up up up up848 up up up up up up up up? up up

Toll Like Receptors

Receptor Kinases

G Protein Coupled Receptors

Cytokine Receptors

Ligands: italic = cAMP; underline = calcium response; bold = phosphoprotein onlyPhosphorylation: up = reproducible increase; up? = possible increase; down = reproducible decrease