Luciferase activity assay was performed using the Luciferase Detection Kit (Promega, Madison, WI, USA), according to the manufacturer's instruction. First, The WT and mutated PLAUR UTR were cloned into PGL4.11-Basic Vector (Promega), producing the vectors PGL4.11-PLAUR-WT-promoter-luc and PGL4.11-PLAUR-mut-promoter-luc MKN45, and 293T cells were co-transfected with different plasmids for 48 h. Finally, the Promega dual-luciferase system (Promega) was used for detection.
Pgl4.11 basic vector
Manufactured by Promega
The PGL4.11-Basic Vector is a plasmid vector used in molecular biology applications. It provides a basic backbone for cloning and expressing genes of interest in various host systems.
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Lab products found in correlation
2 protocols using pgl4.11 basic vector
1
Luciferase Assay for PLAUR UTR Activity
2
Cloning and Mutating miR-148a Promoter
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An 1800-nt fragment of the human miR-148a promoter upstream of the transcription start site was amplified from human genomic DNA. The fragment was digested with NheI-BglII and inserted into pGL4.11 basic vector (Promega) upstream of the luciferase reporter gene. Site-directed mutagenesis was performed on two MITF binding sites at positions 1417 and 841 nucleotides upstream of the transcription start site (primers sequences are given in Table S5 ). pcDNA3-MITF-HA expression vector was a gift from Dr. David. E. Fisher (Harvard Medical School, Boston, MA, USA). pEZX-MT01 3′ UTR MITF luciferase plasmid (GeneCopoeia) was a generous gift from Dr. Dror Avni (Sheba Medical Center, Ramat Gan, Israel).
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