Figure 2 | Oncogene

Figure 2

From: G protein-coupled receptors GPR4 and TDAG8 are oncogenic and overexpressed in human cancers

Figure 2

Activation of multiple signaling pathways by GPR4 and TDAG8. HEK293 cells were co-transfected with the pEF6 plasmid containing cDNAs for either GPR4 or TDAG8, together with one of the four reporter gene plasmids (Stratagene, La Jolla, CA, USA). These reporter gene constructs, pSRE-luc, pCRE-luc, pNFAT-luc, and pFA-CREB/pFR-luc, have firefly luciferase gene under the control of DNA-binding elements for either the Serum Response Factor (in pSRE-luc), cAMP Response Element Binding Protein (CREB) (in pCRE-luc), Nuclear Factor of Activated T cells (in pNFAT-luc), or the trans-reporter system with CREB as the transcription factor (in pFA-CREB/pFR-luc). The CRE-luciferase reporter is from the PathDetect cis-reporting system made by Stratagene. It contains tandem repeats of the cyclic AMP response element (CRE) in the promoter enhancer region of the luciferase reporter gene, which measures transcriptional activation from CRE. The CREB-trans reporter is from the PathDetect CREB Trans-Reporting system by Stratagene. This reporter gene measures more specifically the activation of protein kinase A that phosphorylates and activates CRE-binding protein (CREB). Transfection was done with LipofectAmine 2000 reagent in OPTI-MEM medium (Gibco-BRL, Gaithersberg, MA, USA) in 96-well multiwell plates. At 24 h after transfection, cells grown in OPTI-MEM medium were lysed and luciferase activities were measured on CLIPR (Molecular Devices, Sunnyvale, CA, USA) by using the Luciferase Assay Reagent from Promega (Madison, WI, USA). Transfection efficiency was normalized by co-transfection with the pTK-Renilla luciferase from Promega

Back to article page