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Summary Testing New Cell Lines

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

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Summary Testing New Cell Lines

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  1. SummaryTesting 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. SummaryTesting 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. SummaryTransition 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) Other responses TGF-b c-Fms (M-CSF-R) IL-1b TNF IL6 Il-10 RANKL BNP IFN-g Fcg cAMP responses Ter (b-adrenergic) PGE2 (EP2, EP3, and EP4) VIP PACAP Ca responses MCP-1 (CCR2) C5a (C5a-R) UTP LPA Estrogen/Androgen

  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 3 16 31 J774A.1 3 5 4 2 12 26 AtT-20 4 4 1 2 10 21

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