The Af CS Antibody Lab Eduardo Arteaga Rod
The Af. CS Antibody Lab Eduardo Arteaga Rod Ceja Blythe King
Af. CS Antibody Lab: Year 4 Goals n Transition to RAW 264. 7 macrophage cell line n Double the number of phosphoproteins monitored n Initiate ligand screen for phosphoproteins n n 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 Af. CS Antibody Lab Quantify ligand-induced changes in site specific phosphorylation of selected proteins. n n Aims: sample diversity of cellular response to ligands and identify interactions between ligands Approach: Multiplex Western blotting with mixtures of site specific, phosensitive antibodies Ila Oxendine, Frank Amador, and Jeff Scales manage to smile in the cold room
RAW Cell Ligand Screen Robert Hsueh’s Time Line RAW 264. 7 2003 Af. CS Annual Meeting Robert Hsueh Cell Lab Initial Ligand List/ Response Summary 40 Frozen Chromostocks somes Heping Han Antibody Lab Ligand Screen Results Final Ligand List Dual Ligand Screen Ab Lab: Testing antibodies and ligands 11 phosphoproteins: 80 samples/wk Time courses Vary ligand conc. 05/04 04/04 03/04 02/04 01/04 12/03 11/03 10/03 09/03 08/03 07/03 06/03 05/03 04/03 characterization 1/2 21 phosphoproteins: 170 samples/wk
Results of Antibody Testing and Scoring Are Recorded in the Antibody Database The Af. CS Antibody Database is a resource for the research community to learn how well commercially available antibodies performed in our lab Af. CS 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)* Becky Fulin presents poster #20 Tues. AM: Heping Han will give brief presentation on database
* Phospecific Antibody Targets * * *d/q, m * p 40 phox * * Ribo S 6 p 90 RSK * * Ribo S 6
Toll-like Receptor Signaling * * Phospecific Antibody Targets
Targets of Current Phospecific Antibody Mixtures for Multi-Plex Western Blotting n Mix 1 n n n p 90 RSK (S 380) ERKs (T 202/Y 204) Ribosomal S 6 (S 235/236) Akt (S 473) Mix 2 n n n n PKCm (S 916) STAT 3 (Y 705) STAT 1 a/β (Y 701) NFKB p 65 (S 536) JNKs (T 183/Y 185) p 38 MAPK (T 180/Y 182) Mix 3 n STAT 5 (Y 694) n Ezrin/radixin/moesin n n (T 567/T 564/T 558) GSK 3 a/β (S 21/9) Mix 4 n PKC d/f (S 643/676) n Smad 2 (S 465/467) n p 40 Phox (T 154) Total: 16 Antibodies for 21 phosphoproteins (counting resolvable isoforms)
How Phosphoprotein Data Are Processed for the Ligand Screen Multiplex western blot Image analysis / quantification Export to Bob Sinkovits of the Bioinformatics Group in San Diego for graphing & display
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
RAW 264. 7 Cells: Statistically Significant Phosphoprotein Responses to Single Ligands p < 0. 05 Madhu Natarajan Increased phosphorylation at any time point (1, 3, 10, or 30 min) Decreased phosphorylation Decreased followed by increased phosphorylation
PAM 3 CSK 4, a Synthetic TLR Ligand, Inhibits Interferon -stimulated Phosphorylation of STAT 3 but not STAT 1 IFB P 3 C+IFB Red = Interferon alone, stimulates phosphorylation of STATs 1 & 3 Blue = combination of two ligands Green = P 3 C alone (not seen if STAT band invisible, i. e. STAT 3) PAM 3 CSK 4 binds to heterodimer of TLR 1 & 2
Lipopolysaccharide Inhibits Interferon -stimulated Phosphorylation of STATs 1, 3, & 5 LPS is agonist of TLR 4 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
Summary of Negative Interaction between Interferons and TLR Ligands Inhibition of Interferon - or -stimulated Phosphorylation of STATs Ligand Receptor Inhibition of STAT Phosphorylation LPS TLR 4 STAT 1, 3, & 5 PAM 2 CSK 4 (P 2 C) TLR 2/6 STAT 1 & 3, not 5 PAM 3 CSK 4 (P 3 C) TLR 2/1 STAT 3, not 1 or 5 R 848 TLR 7 STAT 1 & 3
Comparison of Results between the Various Ligand Screen Assays Ligand Pair P-STATs Cytokines c. AMP Calcium n Phosphorylation of STATs Positive TLR ligand Negative No response alone have nointeraction effect on phosphorylation of STATs + IFn TLR or ligands interaction single ligands Single ligands n n TLR ligands selectively inhibit interferon-stimulated phosphorylation of STATs Cytokines n n Interferon or alone has no effect on secretion of cytokines Interferon enhances TLR-stimulated release of cytokines (next slide)
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
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 n n n Ligand Screen: complete three replicates of each ligand pair FXM: assay responses in knockdowns, other perturbations Collaborate with Bio-Rad and Cell Signaling Technology n n n 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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