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GFP PLCδ δ PH domain – membrane to cytoplasm redistribution assay Phospholipase C plays a key role in signal transduction processes for many cell surface receptors Generates DAG and IP3 as second messengers GFP is fused to the Pleckstrin Homology (PH) domain of PLC. This binds to phosphatidylinositol lipids (specifically PIP2). This fusion protein translocates like native PLC Generally applicable to all areas because many agonists that activate signal transduction pathways stimulate PLC delta GFP-PLCδ δ PH domain membrane to cytoplasm redistribution assay Agonist, 5-10 sec Un-stimulated cell: majority of GFP-PLCδ localized on inner surface of plasma membrane. 20-40 sec Stimulated cell: GFP-PLCδ redistributes transiently to cytoplasmic space, Post-stimulation cell: GFP-PLCδ re-accumilates at plasma membrane GFP-PLCδ PH domain Translocation index - (% D peak) ATP Dose Response curve 40 30 20 10 0 -7 -6 -5 -4 ATP conc. (M) Image acquisition & analysis: IN Cell Analyzer 3000 -3 GFP Translocation Assays Assay Response pathways MAPKAP-k2 Rac-1 PLCδ PH domain 2XFYVE domain (domain from Hrs) Example Therapeutic Areas Stress response pathways Inflammation, neuronal growth and differentiation, pain, CNS disorders, ischemia, seizures, skeletal muscle regulation Cytokinesis, phagocytosis, pinocytosis, axon outgrowth, morphogenesis, cellcell contacts, cell polarity, transformation, adhesion, migration Colon and breast cancer, cardiac hypertrophy, myofibrillogenesis, proinflammatory signalling, leukemia, rheumatoid arthritis, progression to AIDS PI(4,5)P2 signalling pathways, adrenoreceptor function, cytoskeletal organization, responses to purinergic agonists, mechanical stress pathways Cardiac diseases/ hypertrophy, cardiac injury, Alzheimer’s disease, bipolar disorders Growth factor (RTK) and cytokine signalling pathways, PI(3)P levels (class III PI3Kinase sensor) Identification of class-specific PI3K inhibitors for development of cancer treatments, immuno-suppressants and anti-inflammatory drugs (Slide 1 of 2) GFP Translocation Assays Response pathways Assay Akt1 Cell survival, proliferation, apoptosis, insulin response pathways Example Therapeutic Areas Cancer (breast, prostate, ovarian, gastric), tumor progression, drug and radiation resistance in cancer therapy, invasion, angiogenesis, diabetes TGF-beta signalling, growth, differentiation, cell survival, regulation of cell proliferation and migration, elaboration of extracellular matrix Oncogenesis, tumor progression, cardiac hypertension, embryonic development, tissue repair, immune function STAT3 Acute-phase response pathways, cytokine signalling, leptin signalling Acute-phase responses, oncogenesis (hematologic, breast, head, neck, prostate cancers), obesity NFAT-c1 Immune responses (IL2, IL4, TNFα gene expression), prostaglandin signalling Immunosuppression, muscle growth SMAD2 (Slide 2 of 2)