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The AfCS Antibody Lab Rod Ceja Blythe King Eduardo Arteaga.

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Presentation on theme: "The AfCS Antibody Lab Rod Ceja Blythe King Eduardo Arteaga."— Presentation transcript:

1 The AfCS Antibody Lab Rod Ceja Blythe King Eduardo Arteaga

2 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

3 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

4 Dual Ligand Screen characterization 04/0305/0306/0307/0308/0309/0310/0311/0312/0301/0402/0403/0404/0405/04 2003 AfCS Annual Meeting 40 Chromo- somes Frozen stocks Initial Ligand List/ Response Summary Final Ligand List Ligand Screen Results RAW 264.7 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

5 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

6 p90 RSK ** * * * * * * * *  * Ribo S6 p40 phox * * Phosphospecific Antibody Targets * Ribo S6 *

7 Toll-like Receptor Signaling * * * * Phosphospecific Antibody Targets

8 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 PKC  (S916) STAT 3 (Y705) STAT 1  /β (Y701) NF  B p65 (S536) JNKs (T183/Y185) p38 MAPK (T180/Y182) Mix 3 STAT 5 (Y694) Ezrin/radixin/moesin (T567/T564/T558) GSK 3  /β (S21/9) Mix 4 PKC  (S643/676) Smad 2 (S465/467) p40 Phox (T154) Total : 16 Antibodies for 21 phosphoproteins (counting resolvable isoforms)

9 Export to Bob Sinkovits of the Bioinformatics Group in San Diego for graphing & display Image analysis / quantification Multiplex western blot How Phosphoprotein Data Are Processed for the Ligand Screen

10 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

11 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

12 PAM3CSK4, a Synthetic TLR Ligand, Inhibits Interferon  - stimulated Phosphorylation of STAT3 but not STAT1 Red = Interferon  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

13 Lipopolysaccharide Inhibits Interferon  -stimulated Phosphorylation of STATs 1, 3, & 5 Red line = Interferon  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

14 Summary of Negative Interaction between Interferons and TLR Ligands LigandReceptorInhibition of STAT Phosphorylation LPSTLR4STAT 1, 3, & 5 PAM2CSK4 (P2C)TLR 2/6STAT 1 & 3, not 5 PAM3CSK4 (P3C)TLR 2/1STAT 3, not 1 or 5 R848TLR 7STAT 1 & 3 Inhibition of Interferon  - or  -stimulated Phosphorylation of STATs

15 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  or  alone has no effect on secretion of cytokines Interferon enhances TLR-stimulated release of cytokines (next slide) Ligand PairP-STATsCytokinescAMPCalcium TLR ligand + IF  or  Negative interaction Positive interaction No response single ligands No response Single ligands

16 Interferon  or  Enhances TLR ligand-induced Secretion of IL-6, IL-10, and RANTES Example: Interferon  enhances LPS-stimulated secretion of IL-4, IL-10, and RANTES

17 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

18 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

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