04 SMumby

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 ScreenRobert 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 Processedfor 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