raw 264.7 two-ligand screen strategy to monitor protein phosphorylation for the macrophage ligand...
Post on 19-Jan-2016
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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
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