HEK293T cells stably expressing full-length EDAR or HaCaT cells were seeded in 12-well dishes one day before transfection. 500 ng of pNF-κB Luc plasmid (Promega, E8491), and 10 ng of pRL‐TK Renilla reference plasmid (Promega, E2231) were co-transfected for each well using Lipofectamine 3000 (Invitrogen). Twenty-four hours after the transfection, an equivalent volume of the supernatant of WT or mutated EDA· A1THD proteins were added to each well, respectively. After 12 h (for HEK293T) or 18 h (for HaCaT), Firefly luciferase activity in the cell lysates was measured and normalized to Renilla luciferase activity using a dual-luciferase reporter assay system (Promega).
Prl tk renilla reference plasmid
Manufactured by Promega
The PRL-TK Renilla reference plasmid is a laboratory tool used as a internal control in reporter assays. It contains the Renilla luciferase gene under the control of the thymidine kinase (TK) promoter, providing a standardized reference signal.
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2 protocols using prl tk renilla reference plasmid
1
Evaluating NF-κB Activation by EDA Proteins
2
Assessing NF-κB Pathway Activation
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Stable HEK293T‐EDAR cells were seeded in 12‐wells plates dishes the day before transfection. Five hundred nanograms of pNF‐κB Luc plasmid (Promega), 10 ng of pRL‐TK Renilla reference plasmid (Promega), and the expression plasmid containing either wild‐type EDA1 or the mutated EDA1 were co‐transfected into each well using Lipofectamine 2000. Twenty‐four hours after the transfection, firefly luciferase activity in the cell lysates was measured and normalized with renilla luciferase activity using the dual luciferase assay system (Promega). Each experiment was performed in triplicate and repeated three times. Data were assessed by Student’s t test (p < .05).
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