04 SMumby

Information about 04 SMumby

Published on November 5, 2007

Author: Julie

Source: authorstream.com

Content

The AfCS Antibody Lab:  The AfCS Antibody Lab Rod Ceja Blythe King Eduardo Arteaga AfCS Antibody Lab: Year 4 Goals:  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:  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 Scales manage to smile in the cold room RAW Cell Ligand Screen Robert Hsueh’s Time Line:  characterization 04/03 05/03 06/03 07/03 08/03 09/03 10/03 11/03 12/03 01/04 02/04 03/04 04/04 05/04 RAW Cell Ligand Screen Robert Hsueh’s Time Line Robert Hsueh Cell Lab Heping Han Antibody Lab Testing antibodies and ligands Vary ligand conc. Time courses Ab Lab: 11 phosphoproteins: 80 samples/wk 21 phosphoproteins: 170 samples/wk 1/2 Results of Antibody Testing and Scoring Are Recorded in the Antibody Database:  AfCS Antibody Database: New Web-based Version Becky 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 #20 Tues. AM: Heping Han will give brief presentation on database Slide6:  p90 RSK * * * * * * * * * *d/q, m * Ribo S6 p40 phox * * Phosphospecific Antibody Targets * Ribo S6 * Slide7:  Toll-like Receptor Signaling * * * * Phosphospecific Antibody Targets Targets of Current Phosphospecific Antibody Mixtures for Multi-Plex Western Blotting:  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) NFKB 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/f (S643/676) Smad 2 (S465/467) p40 Phox (T154) Total: 16 Antibodies for 21 phosphoproteins (counting resolvable isoforms) How Phosphoprotein Data Are Processed for the Ligand Screen:  How Phosphoprotein Data Are Processed for the Ligand Screen Slide10:  Macrophage Ligand Screen: Phosphoproteins Nicholas 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)‡ Ila Oxendine Nick Wong Becky Fulin Jeff Scales RAW 264.7 Cells: Statistically Significant Phosphoprotein Responses to Single Ligands :  RAW 264.7 Cells: Statistically Significant Phosphoprotein Responses to Single Ligands 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:  PAM3CSK4, a Synthetic TLR Ligand, Inhibits Interferon b-stimulated Phosphorylation of STAT3 but not STAT1 Red = Interferon b alone, stimulates phosphorylation of STATs 1 & 3 Blue = 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:  Lipopolysaccharide Inhibits Interferon b-stimulated Phosphorylation of STATs 1, 3, & 5 Red line = Interferon b alone, stimulates phosphorylation of all three STATs Blue 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:  Summary of Negative Interaction between Interferons and TLR Ligands Inhibition of Interferon - or -stimulated Phosphorylation of STATs Comparison of Results between the Various Ligand Screen Assays:  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 Interferon a or b alone has no effect on secretion of cytokines Interferon enhances TLR-stimulated release of cytokines (next slide) Interferon a or b Enhances TLR ligand-induced Secretion of IL-6, IL-10, and RANTES:  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 # 24:  Poster # 24 Macrophage Ligand Screen: Cytokines Ruth Levitz, Robert Hsueh‡, Lonnie Sorrells, Robert Sinkovitz*,Heping Han and Ron Taussig The 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:  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 Zhao on Halloween

Related presentations


Other presentations created by Julie

Traditions and customs
16. 11. 2007
0 views

Traditions and customs

cereal en
04. 10. 2007
0 views

cereal en

WinCE Overview
29. 11. 2007
0 views

WinCE Overview

S1 01 McCurry
01. 12. 2007
0 views

S1 01 McCurry

cootiecatcher gaillovely com
05. 11. 2007
0 views

cootiecatcher gaillovely com

exp meth2
05. 11. 2007
0 views

exp meth2

JDF Color
05. 11. 2007
0 views

JDF Color

Liquid Nitrogen Tank
07. 11. 2007
0 views

Liquid Nitrogen Tank

chapter 1 powerpoint im
12. 11. 2007
0 views

chapter 1 powerpoint im

japonsko
13. 11. 2007
0 views

japonsko

SupplierHandbook June2005
16. 11. 2007
0 views

SupplierHandbook June2005

6 1
19. 11. 2007
0 views

6 1

MolMod C371
21. 11. 2007
0 views

MolMod C371

AdaptationsMustelinae
22. 11. 2007
0 views

AdaptationsMustelinae

6 adsl
26. 11. 2007
0 views

6 adsl

mspo
26. 11. 2007
0 views

mspo

20020103 goad oc ecampus
03. 12. 2007
0 views

20020103 goad oc ecampus

history2
01. 01. 2008
0 views

history2

NZ Ethnobotany
04. 01. 2008
0 views

NZ Ethnobotany

EaES350 10
04. 01. 2008
0 views

EaES350 10

Lesson 16 Early Cold War
19. 12. 2007
0 views

Lesson 16 Early Cold War

MilkingCenterWastewa terOptions
06. 11. 2007
0 views

MilkingCenterWastewa terOptions

file Linda Fosburg
20. 11. 2007
0 views

file Linda Fosburg

303lec11
07. 01. 2008
0 views

303lec11

ai23 lecture4H
04. 01. 2008
0 views

ai23 lecture4H

14 121 1430
01. 12. 2007
0 views

14 121 1430

BienvenidosaMCasa2
05. 11. 2007
0 views

BienvenidosaMCasa2

day05
26. 02. 2008
0 views

day05

Lewis and Clark 2
28. 02. 2008
0 views

Lewis and Clark 2

I OnGuard
04. 03. 2008
0 views

I OnGuard

Cetron
11. 03. 2008
0 views

Cetron

fox sra2002
04. 12. 2007
0 views

fox sra2002

johnh sensys business 071108a
18. 03. 2008
0 views

johnh sensys business 071108a

Rowanrobinson
15. 11. 2007
0 views

Rowanrobinson

FREE ZONES IN UAE
30. 03. 2008
0 views

FREE ZONES IN UAE

12 Dierker JPSI NUW July07
28. 09. 2007
0 views

12 Dierker JPSI NUW July07

BETWEEN THE WARS
13. 04. 2008
0 views

BETWEEN THE WARS

habitat101
03. 01. 2008
0 views

habitat101

NicePotassium
05. 11. 2007
0 views

NicePotassium

Paleozoic
03. 10. 2007
0 views

Paleozoic

7aSchallier
05. 12. 2007
0 views

7aSchallier

ART273LEC1
24. 02. 2008
0 views

ART273LEC1

WGPOA 2007 Annual Mtg Agenda
02. 11. 2007
0 views

WGPOA 2007 Annual Mtg Agenda

atriptoagra
07. 01. 2008
0 views

atriptoagra

10 30 07
17. 12. 2007
0 views

10 30 07

cirtric phased web
31. 12. 2007
0 views

cirtric phased web

pepgrant
23. 11. 2007
0 views

pepgrant

sikkim
07. 01. 2008
0 views

sikkim

background rs
28. 11. 2007
0 views

background rs

NaturalAnalogues2
09. 10. 2007
0 views

NaturalAnalogues2