1. Monitoring Protein Phosphorylation for the Ligand Screens Goal: sample diversity of cellular response to inputs Current Approach: Multiplex Western blotting with mixtures of phosphospecific antibodies Quantify ligand-induced changes in site-specific protein phosphorylation

2. Ligand Screens: B Cells Single ligand screen completed Two phosphospecific antibody mixes for quantification of 11 phosphoproteins Timecourse data on-line Poster: “Analysis of B Cell Single Ligand Data” Robert Sinkovits and Dennis Mock Dual ligand screen experiments and analysis in progress. See posters: “Dual Ligand Screen in Splenic B Cells” Keng-Mean Lin, Madhusudan Natarajan, Robert Hsueh, and Heping Han “Dual Ligand Screen in Splenic B Cells (continued)” Keng-Mean Lin, Madhusudan Natarajan, Robert Hsueh, and Heping Han

3. Ligand Screens: WEHI-231 Poised to initiate screen: characterization of cells, derivation of protocols, validation of phosphospecific antibodies completed See poster: “Preparation and Characterization of WEHI-231 for the Ligand Screens” Robert Hsueh, Keng-Mean Lin, and Heping Han

4. Ligand Screens: Cardiac Myocytes Two new antibody mixes developed Some antibodies that worked well for B cells did not for myocytes Added antibodies for myocyte specific phosphoproteins Ligand dose determinations completed Dual ligand screen initiated. See posters: “Isolation and Culture of Adult Mouse Cardiac Myocytes for Signaling Studies” Timothy D. O'Connell, Yan G. Ni, Keng-Mean Lin, Heping Han, and Zhen Yan “Ligand Screen in Mouse Adult Cardiac Myocytes” Yan G. Ni, Keng-Mean Lin, Heping Han, and Timothy D. O'Connell

5. Current Phosphospecific Antibody Mixtures B cellsMyocytes Mix 1Mix 2Mix 3Mix 4 STAT6STAT3 STAT5 p90RSKPKC μAktp70S6 Kinase AktNFkB p65GSK 3α/βERK JNKp38 MAPKRibosomal S6 p38 MAPKTroponin IMyosin L Chain IkBαPhospholamban Each antibody is directed to a specific site of phosphorylation on protein Color-coding highlights 8 different pathways currently monitored in each cell type Prospective: Can we assay more phosphoproteins? Add ribosomal S6 and myosin light chain to Mix 1 Another mix for leukocytes: Could start with p70 S6 kinase and GSK 3 from Mix 3 & 4

6. Insulin Receptor Signaling   *  *   * * Antibody leukocyte mix * Antibody in myocyte mix * Good antibody not in mix yet 

7. Antibody Database –Tabulation of results from antibody testing by Western immunoblotting –Now available on-line (go to Data Center, Resources) –Easy to spot antibodies that yielded promising results (highlighted with red check marks) –See poster: “AfCS Antibody Laboratory’s Database: Tabulation of Our Experience with Commercially Available Antibodies” Heping Han, Becky Fulin, Lonnie Sorrells, Ruth Levitz, and Susanne Mumby

8. Monitoring Protein Phosphorylation: Increasing Our Repertoire 1 of 7 phosphospecific antibodies we test is suitable for multiplex Western blotting of whole cell lysates Additional promising antibodies to proteins in pathways not represented by antibody Mixes 1 & 2 Smad 2 (S465/467) STAT1 (Y701) PKC delta (T505) Total current possibilities for multiplex = 10 Capacity: 80 samples/week with two mixes Best case scenario 2 more antibody mixtures for multiplex Western blotting Doubles the gels+blots, decreases number of samples we can assay to 40/week Additional antibodies not good enough to multiplex More cells required More gels/blots to process means less than 40 samples/week An alternative, less labor intensive assay could be our salvation

9. Monitoring More Phosphoproteins: Alternative Assay Multiplex fluorescent bead technology Potential for monitoring 100 different analytes per well of 96-well plate Licensed by Luminex Bio-Rad’s system: Bio-Plex Cytokines Phosphoproteins AfCS and Bio-Rad collaborate on development and validation of phosphoprotein assays

10. Bio-Plex Phosphoprotein Assays

11. Three-way Collaboration 1. AfCS and Cell Signaling Technology AfCS suggests targets for novel antibodies CST produces peptide antigen and antibodies CST and AfCS test antibodies AfCS uses antibodies and CST sells them 2. CST and Bio-Rad Collaboration agreement pending Development of antibody pairs (capture and phosphospecific) for Bio-Plex 3. Bio-Rad and AfCS Develop/validate antibody pairs for Bio-Plex Set up Bio-Plex in AfCS to monitor protein phosphorylation

12. Benefits of 3-way Collaboration AfCS: Increase number of phosphoproteins monitored = more data for modeling effort No increase in number of cells required CST and Bio-Rad: Company name and product exposure Increased sales Signaling Community: More AfCS data available to draw from Increased number of validated antibody reagents available on the market Demonstration of the utility of Luminex technology for analysis of phosphoproteins