Tranduction Regulation of Transcription

Add ligand or inhibitor isolate total rna from cells

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Add ligand or inhibitor Isolate total RNA from cells Perform reverse transcription to generate cDNA Do PCR to determine the presence of a specific gene Compare amount of specific transcript in control sample to test sample Normalize the amount of total starting material to the expression of “housekeeping” genes (Genes that don’t change in response to pathway perturbation such as beta-actin, GAPDH, HPRT) Detection of pathway activity by RT-PCR for target genes AAAAA TTTTT 9 AAAAA ! # $ % ’( )*+,)-+.) /012+), /012+), 3 456 Gene expression (relative to GAPDH)
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Plasmid with reporter Fluorescence Colorimetric Luminescence Clone pathway specific response element upstream of reporter DNA sequence that R-Smad binds to in TGF-beta/BMP target genes Transfect response element- reporter construct and transfection control plasmid into cells in culture Activate or repress pathway Add ligand or inhibitor Add substrate for luciferase Measure amount of luciferase made as surrogate for pathway activity Detection of pathway activity by luciferase reporter assay 10
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11 Receptor Tyrosine Kinase Signaling Players Receptors - Tyrosine Kinases Ligands - Growth Factors Intracellular Effectors - Grb2 Adaptor Sos GEF Ras G-protein Raf Kinase MAP Kinases Cell type-specific transcriptional regulators 17-25% of all solid tumors harbor activating mutations in kras
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Receptor Tyrosine Kinases Dimers of single-pass transmembrane proteins Extracellular portion binds ligands (often growth factors) Cytosolic portion has Tyrosine Kinase activity Trans-autophosphorylate tyrosine residues on opposite dimer 12
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GF binds and induces conformational change in receptor Receptor dimerizes and becomes activated Active receptor transphosphorylates tyrosine residue on opposite receptor Phosphotyrosine on receptor act as dock for SH2 containing protein, such as Grb2 Events following GF binding 13
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Phosphotyrosines on RTK serve as docking sites for effector molecules Src homology domain 2 (SH2) -binds to phosphotyrosines -specific phosphotyrosine dictated by sequence surrounding Tyr amino acid
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Add ligand or inhibitor Isolate total RNA from cells...

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