The AfCS Antibody Lab

1. The AfCS Antibody Lab Eduardo Arteaga Rod Ceja Blythe King

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 insite 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. RAW 264.7 Initial Ligand List/ Response Summary 40 Chromo- somes 2003 AfCS Annual Meeting Ligand Screen Results Final Ligand List Frozen stocks Dual Ligand Screen RAW Cell Ligand ScreenRobert Hsueh’s Time Line Heping Han Antibody Lab Robert Hsueh Cell Lab 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 Time courses Ab Lab: Testing antibodies and ligands 1/2 11 phosphoproteins: 80 samples/wk Vary ligand conc. 21 phosphoproteins: 170 samples/wk

5. 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 AfCS Antibody Database: New Web-based VersionBecky 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)* Becky Fulin presents poster #20 Tues. AM: Heping Han will give brief presentation on database

6. * Phosphospecific Antibody Targets * * * * * * * * p90 RSK * Ribo S6 *d/q, m * * * Ribo S6 * p40 phox

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

8. 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 PKCd/f (S643/676) Smad 2 (S465/467) p40 Phox (T154) Targets of Current Phosphospecific Antibody Mixtures for Multi-Plex Western Blotting Total: 16 Antibodies for 21 phosphoproteins (counting resolvable isoforms)

9. Multiplex western blot Image analysis / quantification Export to Bob Sinkovits of the Bioinformatics Group in San Diego for graphing & display How Phosphoprotein Data Are Processedfor 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)‡ Becky Fulin Nick Wong Ila Oxendine 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) p < 0.05 Decreased phosphorylation Decreased followed by increased phosphorylation Madhu Natarajan

12. PAM3CSK4, a Synthetic TLR Ligand, Inhibits Interferon b-stimulated Phosphorylation of STAT3 but not STAT1 IFB P3C P3C+IFB 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) PAM3CSK4 binds to heterodimer of TLR 1 & 2

13. Lipopolysaccharide Inhibits Interferon b-stimulated Phosphorylation of STATs 1, 3, & 5 LPS is agonist of TLR 4 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

14. Summary of Negative Interaction between Interferons and TLR Ligands 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 a or b alone has no effect on secretion of cytokines • Interferon enhances TLR-stimulated release of cytokines (next slide)

16. 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

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