1. Summary Testing New Cell Lines Currently underway: exploration of 7 cell lines (J774A.1, Raw 264.7, AtT-20, 3T3-L1, IC21, Neuro 2a, N1E-115) “A” list: J774A.1, Raw 264.7, AtT-20 Rich responses (inputs and outputs) Ectopic expression of proteins 80-90% by lentivirus in Macrophage lines 20-50% by transfection (Raw 264.7 cells, J774A.1 poorer) RNAi works (in macrophage lines)

2. Summary Testing New Cell Lines Preliminary assessment of AfCS assays Second messenger responses (Ca, cAMP) verified Current antibody cocktails are ready for use in macrophage lines Macrophage lines suitable for microscopy Anticipate no problems with lipid analysis MA analysis should not be problematic Protein trap – Expression levels higher than WEHI cells

3. Summary Transition from Current Focus Higher throughput anticipated Cultured cells vs isolation of primary cells from mice Delays Characterization of cell line stability New libraries for Yeast 2 hybrid Development of new assays (chemotaxis, phagocytosis, secretion, etc.) Data handling/display (transparent) Ligand list

4. Ligand List RAW264.7 (Literature) cAMP responses Ter  -adrenergic) PGE2 (EP2, EP3, and EP4) VIP PACAP Ca responses MCP-1 (CCR2) C5a (C5a-R) UTP LPA Estrogen/Androgen Other responses TGF-  c-Fms (M-CSF-R) IL-1  TNF IL6 Il-10 RANKL BNP IFN-  Fc 

5. GPCR List RAW264.7 (MA analysis) Adenosine A2b Adrenomedulin GRPbombesin Cannabinoid 1 Chemokine C-C 2 (Cmkbr2) Chemokine CCX CKR Chemokine C-X-C 3 (Cmkar3) Chemokine C-X3-C R1 (similar to) Cysteinyl leukotriene 1 Dopamine 3 Endothelin B Estrogen LPA (vzg-1) LPA 2 Malanocortin 5 Nerotensin 2 (B) NPY –Y1 PACAP 1 PGE 1 PGE EP2 P2Y 4 P2Y5 P2Y 12 PGE 4 5-HT 1B 5-HT 2C Somatostatin 2 SDF1 Stromal Cell Derived factor 2 V1a arginine vasopressin

6. Ligand List for Cell Lines on “A List” Pathway Gs Gi Gq Other GPCR Other Rec. Toll-like, Cytokine, Tyr Kinase, Adh, other Total Raw 264.7 5 4 3 16 31 J774A.1 3 5 4 2 12 26 AtT-20 4 1 2 10 21