HEK293 cells (ATCC CRL-1573) were obtained from ATCC and were grown in a 48-well tissue culture plates in DMEM containing 10% FBS until ~40% of confluency was reached. HEK293 cells were transfected with a total of 250 ng of plasmid DNA per well, consisting of 25 ng of the reporter construct pNF-κB-luc, 25 ng of the normalization vector pTK-LacZ, and 200 ng of the different combinations of mammalian expression vectors carrying the indicated gene (empty control vector, pCMV-HA-VceC2 , pCMV-HA-TRAF2DN (this study), hNOD1-3xFlag, hNOD2-3xFlag, pCMV-HA-hRip2, hNOD1DN-3xFlag, hNOD2DN-3xFlag or pCMV-HA-Rip2DN3 and pCMV-myc-CDC42DN4 . The dominant-negative form of TRAF2, lacking an amino-terminal RING finger domain5 , was PCR amplified from cDNA prepared from HEK293 cells and cloned into the mammalian expression vector pCMV-HA (BD Biosciences Clontech). Forty-eight hours after transfection, cells were lysed either without any treatment, or stimulated with C12-iE-DAP (1000 ng/mL, InvivoGen) and MDP (10 µg/mL, InvivoGen). After five hours of treatment the cells were lysed and analysed for β-galactosidase and luciferase activity (Promega). FuGene HD (Roche) was used as a transfection reagent according to the manufacturer’s instructions. Cell lines were monitored for Mycoplasma contamination.
β galactosidase and luciferase activity
Manufactured by Promega
β-galactosidase and luciferase are enzymes commonly used as reporter genes in molecular biology experiments. β-galactosidase catalyzes the hydrolysis of the disaccharide lactose into glucose and galactose. Luciferase catalyzes the oxidation of luciferin, producing light. These enzymes are useful for monitoring gene expression and various cellular processes.
Automatically generated - may contain errors
2 protocols using β galactosidase and luciferase activity
1
NOD1/NOD2 Signaling Pathway Modulation
2
NOD1/NOD2 Signaling Pathway Modulation
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HEK293 cells (ATCC CRL-1573) were obtained from ATCC and were grown in a 48-well tissue culture plates in DMEM containing 10% FBS until ~40% of confluency was reached. HEK293 cells were transfected with a total of 250 ng of plasmid DNA per well, consisting of 25 ng of the reporter construct pNF-κB-luc, 25 ng of the normalization vector pTK-LacZ, and 200 ng of the different combinations of mammalian expression vectors carrying the indicated gene (empty control vector, pCMV-HA-VceC2 , pCMV-HA-TRAF2DN (this study), hNOD1-3xFlag, hNOD2-3xFlag, pCMV-HA-hRip2, hNOD1DN-3xFlag, hNOD2DN-3xFlag or pCMV-HA-Rip2DN3 and pCMV-myc-CDC42DN4 . The dominant-negative form of TRAF2, lacking an amino-terminal RING finger domain5 , was PCR amplified from cDNA prepared from HEK293 cells and cloned into the mammalian expression vector pCMV-HA (BD Biosciences Clontech). Forty-eight hours after transfection, cells were lysed either without any treatment, or stimulated with C12-iE-DAP (1000 ng/mL, InvivoGen) and MDP (10 µg/mL, InvivoGen). After five hours of treatment the cells were lysed and analysed for β-galactosidase and luciferase activity (Promega). FuGene HD (Roche) was used as a transfection reagent according to the manufacturer’s instructions. Cell lines were monitored for Mycoplasma contamination.
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