Pgl3 basic firefly luciferase expression vector

The Renilla luciferase-reporter plasmids containing human IQGAP1 mRNA [44 (link)] with either wild-type (Wt; #14503) or mutant (Mut; #14504) miR-124 binding sites (875–882 bp and 1172–1178 bp from the start site of the 3′-UTR) were obtained from Addgene (Cambridge, MA, USA). Wt or Mut IQGAP1 reporter vectors, together with the pGL3-basic firefly luciferase expression vector as a reference control (Promega, San Luis Obispo, CA, USA), were transfected with 30 nM of the miR-124 mimic or inhibitor using Lipofectamine 2000 (Invitrogen, CA, USA). The Renilla and firefly luciferase activities were measured using the Dual Luciferase assay kit (Promega, WI, USA) 24 hours after transfection. The Renilla luciferase activity was normalized to the firefly luciferase activity.

The Transcription Factor Binding Site Prediction (TFBS)-JASPAR database (http://JASPAR.genereg.net/, accessed on 23 November 2021) and ALGGEN-PROMO (http://alggen.lsi.upc.es, accessed on 23 November 2021) were used to search for potential binding sites to determine the potential function of the PmDsx binding site on the core PmIAG promoter. The full-length ORF of PmDsx was amplified and inserted into the pCMV-C-myc vector (Beyotime, China) containing the Hind III/Bgl I site, called pCMV-PmDsx. In addition, four truncated mutants from the PmIAG promoter were designed based on the location of the PmDsx predicted binding site. These fragments have a common 3’-end at −3 bp (translation start codon defined as + 1) and 5’-ends at −1629, −1193, −938, and −371 were amplified using specific primers (Table 1). The vectors were named pGL3-basic-Luc1, pGL3-basic-Luc2, pGL3-basic-Luc3, and pGL3-basic-Luc4, and each fragment was cloned into the pGL3-Basic firefly luciferase expression vector (Promega, USA) for the dual-luciferase reporter assay. The pRL-TK luciferase reporter vector was considered an internal control.