pGL4 Signaling Vectors

High levels of reporter induction for Nuclear Factor of Activated T-cells (NFAT) Response Element containing vector in transient transfections. pGL4.30 Vector was transiently transfected into HEK293 cells. After 24 hours, reporter gene expression was induced by adding 1μM ionomycin plus 10ng/ml PMA for 17 hours. Noninduced controls were treated with an equivalent amount of DMSO. Luciferase activity was measured using the Bright-Glo™ Luciferase Assay System (Cat.# E2610). Results will vary depending on the cell line and the transfection protocol used.

High levels of reporter induction for Serum Response Element (SRE) containing vector in transient transfections. pGL4.33 Vector was transiently transfected into HEK293 cells. Four hours after transfection, cells were changed into serum-starved media. After 24 hours following transfection, reporter gene expression was induced 20% FBS plus 10ng/ml PMA for 6 hours. Noninduced controls were treated with an equivalent amount of standard media. Luciferase activity was measured using the ONE-Glo™ Luciferase Assay System (Cat.# E6110). Results will vary depending on the cell line and the transfection protocol used.

Representative data for pGL4.37[luc2P/ARE/Hygro] in HEK293 cells upon stimulation with tBHQ or D,L-Sulforaphane. HEK293 cells were transiently transfected with pGL4.37[luc2P/ARE/Hygro] and pGL4.75 and assayed in 96-well format after 18 hours stimulation with tBHQ or D,L-Sulforaphane as described. Firefly luciferase luminescence normalized to the Renilla luciferase control is shown, with error bars indicating the S.E.M. for six replicates. Luminescence was detected after addition of Dual-Glo® reagents, using a GloMax® 96 instrument with a 0.5 second integration time.

Representative data for pGL4.39[luc2P/ATF6 RE/Hygro] in HeLa cells upon stimulation with tunicamycin. HeLa cells were transiently transfected with pGL4.39[luc2P/ATF6 RE/Hygro] and pGL4.75 and assayed in 96-well format after 18 hours stimulation with tunicamycin as indicated in the protocol. Firefly luciferase luminescence normalized to the Renilla luciferase control is shown, with error bars indicating the S.E.M. for six replicates. Luminescence was detected after addition of Dual-Glo® reagents, using a GloMax® 96 instrument with a 0.5 second integration time.

Representative data for pGL4.41[luc2P/HSE/Hygro] in HepG2 cells upon stimulation with 17-AAG or CdCl2. HepG2 cells were transiently transfected with pGL4.41[luc2P/HSE/Hygro] and pGL4.75 and assayed in 96-well format after six hours stimulation with 17-AAG or CdCl2 as indicated. Firefly luciferase luminescence normalized to the Renilla luciferase control is shown. Error bars indicate the S.E.M. for six replicates. Luminescence was detected after addition of Dual-Glo® reagents, using a GloMax® 96 instrument with a 0.5 second integration time.

Representative data for pGL4.43[luc2P/XRE/Hygro] in HepG2 cells upon stimulation with TCDD. HepG2 cells were transiently transfected with pGL4.43[luc2P/XRE/Hygro] and pGL4.75 and assayed in 96-well format after 24 hours stimulation with TCDD as indicated in the protocol. Firefly luciferase luminescence normalized to the Renilla luciferase control is shown, with error bars indicating the S.E.M. for six replicates. Luminescence was detected after addition of Dual-Glo® reagents, using a GloMax® 96 instrument with a 0.5 second integration time.

Representative data for pGL4.45[luc2P/ISRE/Hygro] in U2OS cells upon stimulation with IFNα. U2OS cells were transiently transfected with pGL4.45[luc2P/ISRE/Hygro] and assayed in 96-well format after 16 hours stimulation with IFNα as indicated in the protocol. Firefly luciferase luminescence normalized to untreated cells is shown, with error bars indicating the S.E.M. for three replicates. Luminescence was detected after addition of ONE-Glo® reagent, using a GloMax® Multi+ instrument with a 0.5 second integration time.

Representative data for pGL4.52[luc2P/STAT5 RE/Hygro] in Ba/F3 cells upon stimulation with mIL-3. Ba/F3 cells were transiently transfected with pGL4.52[luc2P/STAT5 RE/Hygro] Vector and assayed in 96-well format after 4 hours stimulation with mIL-3 as indicated in the protocol. Firefly luciferase luminescence normalized to untreated cells is shown, with error bars indicating the S.E.M. for four replicates. Luminescence was detected after addition of ONE-Glo® reagent, using a GloMax® Multi+ instrument with a 0.5-second integration time.

Representative data for pGL4.49[luc2P/TCF-LEF RE/Hygro] in HEK293 cells upon stimulation with mWnt3a or LiCl. HEK293 cells were transiently transfected with pGL4.49[luc2P/TCF-LEF RE/Hygro] and assayed in 96-well format after 8 hours stimulation with mWnt3a or LiCl as indicated in the protocol. Firefly luciferase luminescence normalized to untreated cells is shown, with error bars indicating S.E.M. for three replicates. Luminescence was detected after addition of ONE-Glo™ reagent, using a GloMax® 96 instrument with a 0.5 second integration time.