CD47 LightSwitch Promoter Reporter GoClones (RenSP, S710450), Empty promoter vectors (S790005) and Cypridina TK Control constructs (pTK-Cluc, SN0322S) were obtained from SwitchGear Genomics and transfected into HEK cells using Lipofectamine 2000 (Invitrogen). For overexpression assays, expression plasmids for NFKB1 (p50), RELA (p65), NFKB2 (p52), and c-Rel were obtained from Addgene (#21965, #21966, #23289, #27256, respectively). Empty vector pCMV4 was generated by Hind III digestion of #21966, and pcDNA3.1 was obtained from life technologies. 50 ng of plasmid was co-transfected with 45ng of the RenSP reporter and 5ng of the pTK-Cluc reporter construct. Media was changed to fresh DMEM and 50 ng/ml of TNF-α was added 2, 8, 24, and 36 hours prior to harvest. The cell lysate and supernatant were harvested 48 hours after transfection and dual luciferase activity was measured with the LightSwitch Dual Assay System using a SpectraMax L luminometer (Molecular Devices), according to the manufacturer’s instructions. Relative luciferase activity (Renilla/Cypridina luciferase ratio) was quantified as the percentage change relative to the basal values obtained from control-transfected cells not exposed to TNF-α treatment.
Cd47 lightswitch promoter reporter goclones
Manufactured by SwitchGear Genomics
The CD47 LightSwitch Promoter Reporter GoClones is a pre-constructed reporter gene system that allows for the analysis of CD47 promoter activity. It includes a vector containing the CD47 promoter sequence upstream of a luciferase reporter gene.
Automatically generated - may contain errors
2 protocols using cd47 lightswitch promoter reporter goclones
1
Transcriptional Regulation of NF-κB Pathway
2
Transcriptional Regulation of NF-κB Pathway
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CD47 LightSwitch Promoter Reporter GoClones (RenSP, S710450), Empty promoter vectors (S790005) and Cypridina TK Control constructs (pTK-Cluc, SN0322S) were obtained from SwitchGear Genomics and transfected into HEK cells using Lipofectamine 2000 (Invitrogen). For overexpression assays, expression plasmids for NFKB1 (p50), RELA (p65), NFKB2 (p52), and c-Rel were obtained from Addgene (#21965, #21966, #23289, #27256, respectively). Empty vector pCMV4 was generated by Hind III digestion of #21966, and pcDNA3.1 was obtained from life technologies. 50 ng of plasmid was co-transfected with 45ng of the RenSP reporter and 5ng of the pTK-Cluc reporter construct. Media was changed to fresh DMEM and 50 ng/ml of TNF-α was added 2, 8, 24, and 36 hours prior to harvest. The cell lysate and supernatant were harvested 48 hours after transfection and dual luciferase activity was measured with the LightSwitch Dual Assay System using a SpectraMax L luminometer (Molecular Devices), according to the manufacturer’s instructions. Relative luciferase activity (Renilla/Cypridina luciferase ratio) was quantified as the percentage change relative to the basal values obtained from control-transfected cells not exposed to TNF-α treatment.
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