1. RAW 264.7 two-ligand screen Strategy to Monitor Protein Phosphorylation for the Macrophage Ligand Screen  Cell Preparation and Analysis Lab: Expose RAW 264.7 cells to single and paired combinations of ligands –Goal: ~23 ligands and their ~253 paired combinations: conduct each 3 times on different days –Experimental design: Expose cells to 4 single ligands and their 6 possible combinations 4 time points: 1’, 3’, 10’, 30’ Extract cells with SDS-PAGE sample buffer (supplemented with inhibitors of proteases and phosphatases) including 3 cultures of untreated cells = 43 samples per experiment  Antibody Lab: Assay 21 phosphoproteins by multiplex Western blotting –Centrifuge cell lysates to remove viscous DNA –Produce blots with quadruplicate pairs of Bio-Rad Criterion gel (26-well, 4-20% gradient) –Process each pair of blots with one of 4 mixtures of site-specific, phosphosensitive antibodies –Create images of blots with Storm 860 imager –Quantify signal from 21 phosphoproteins with aid of ImageMaster 1D Elite software –Transmit data to Bioinformatics Lab  Bioinformatics Lab: Process data –Create time course graphs from phosphoprotein data –Display graphs on web: www.signaling-gateway.org P2C + MCF: Thumbnail Graphs (9 of 21 shown) Click Each thumbnail links to a page displaying all independent experiments for these conditions For example, click on Akt to see its 4 replicates Replicate Time Courses of Akt Phosphorylation in Response to MCF + P2C Phosphorylation of Akt reveals synergy of MCF and P2C in 3 of 4 independent experiments Export to Bioinformatics Group in San Diego for graphing & display How Ligand Screen Data Are Processed Image analysis / quantification Multiplex western blot Single Ligand: P2C Thumbnail Display Time courses of 21 phosphoproteins are displayed for representative experiment Click on one time course, such as p38 MAPK, and you will be taken to display of replicate experiments for this phosphoprotein 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) ‡ DATA CENTER The AfCS Antibody Laboratory: Francisco Amador, Eduardo Arteaga, Rodrigo Ceja, Becky Fulin, Blythe King, Ila Oxendine, Jeff Scales, and Susanne Mumby (Director); The AfCS Cell Preparation and Analysis Laboratory: Julie Collins, Richard Davis, Katherine Hawes, Jason Polasek, Amy Pope, Meghdad Rahdar, Melissa Stalder, Audra Wendt, and Paul Sternweis (Director); Data Manager for the AfCS Laboratories at UT Southwestern: Lonnie Sorrells Associate Director of the AfCS: Ron Taussig Acknowledgements Abstract A major focus of the AfCS Antibody Laboratory is to quantify ligand-induced changes in site-specific phosphorylation of chosen signaling proteins. This is one approach to defining interactions between pathways, which will contribute to the development of a model for cell signaling. The AfCS Cell Preparation and Analysis Laboratory produces extracts of RAW.264.7 cells that have been exposed to 23 receptor ligands, both alone and in paired combinations. The Antibody Lab assays protein phosphorylation in these samples by multiplex Western blotting with four mixtures of site-specific, phosphosensitive antibodies. The Antibody Laboratory quantifies the signal for 21 phosphoproteins and transmits the data to the Bioinformatics Laboratory, which in turn produces graphs of the time courses (of fold change in phosphorylation) and displays them on the web. There are essentially two ways to view the phosphoprotein data on the AfCS website (www.signaling-gateway.org, >> Data Center). Accessing the ‘RAW 264.7 ligand screen’ page retrieves a summary table listing the ligands tested and links to the data from each ligand acting alone. The ‘RAW 264.7 two-ligand screen’ page displays a 2-dimensional matrix with links to time courses of phosphoprotein responses to each paired combination of ligands viewed for all 21 phosphoproteins from a single representative experiment (as “thumbnail” sketches), which in turn is linked to a page that shows results from all replicate experiments that have been conducted with a chosen ligand pair. Single ligand time courses culled from double ligand screen experiments Replicates of p38 Phosphorylation in Response to P2C (4 of 7 time courses shown here) Summary of Phosphoprotein Responses to Single Ligands All mixtures of phosphospecific antibodies include a conventional antibody against Rho GDI (not listed). We use the signal for Rho GDI (a 28 kDa protein not anticipated to change significantly during 30 min assays) to normalize for lane-to-lane variability in total protein load. RAW 264.7 ligand screen click Example: click on MCF + P2C (conducted 4 times) Double Ligand Screen Matrix from AfCS Data Center: each D links to data for corresponding two-ligand interaction

2. Ligands: italic = cAMP; underline = calcium response; bold = phosphoprotein only Phosphorylation: up = reproducible increase; up? = possible increase; down = reproducible decrease