Pgl3 basic pgl3b

According to the results of CHIP, four, one and five putative KBSs, respectively, were predicted from the promoter regions of human BCL2 (positions −1201 to −1), BAX (positions −645 to −317) and BIK (positions −1200 to −1). The above promoter regions were amplified from genomic DNA and were subcloned into firefly luciferase reporter vector pGL3-Basic (pGL3b) or pGL4.1 (Promega, Madison, WI) to get pGL3b-BCL2, pGL4.1-BAX and pGL4.1-BIK. Constructs with mutations of the putative KBSs were generated using mutagenic oligonucleotide primers according to the manual of the GeneTailor Site-Directed Mutagenesis System (Invitrogen). HUVECs were cotransfected with firefly luciferase reporter constructs and Renilla luciferase reporter vector pRL-TK (E2241, Promega) together with pCDNA3.1-Kaiso or pCDNA3.1 empty vector using FuGENE HD Transfection Reagent (Roche, Mannheim, Germany). Then, 48 h after transfection, relative luciferase activity represented as the ratio of firefly to Renilla was measured with a Dual-Luciferase Reporter Assay System (Promega). All primers used for genomic DNA amplification and site-directed mutation are listed in Supplementary Tables S4–S5.

CACNA1C reporter gene constructs were generated by PCR amplification of the promoter region spanning the transcriptional start site which is denoted by +1; P1 (−149 to +201), P2 (−399 to +201) P3 (−838 to +201). Fragments were amplified from SH-SY5Y DNA using KOD Extreme hot start polymerase (Merck Millipore). The PCR products were subsequently cloned into the pGL3-basic (pGL3b) (Promega) luciferase reporter gene vector using Gibson Isothermal Assembly cloning kit (New England Biolabs). Primer sets and expected product sizes are described in Supplementary Table 1. The constructs were transformed into XL-10 Gold ultracompetent cells (Agilent Technologies). All constructs were confirmed by sequencing (Source Bioscience).

The CFAP410 VNTR was amplified from genomic DNA by PCR using the KOD Hot Start DNA polymerase following the recommended protocol. The VNTR was subcloned into the intermediate Zero Blunt PCR Vector (Invitrogen). This intermediate plasmid was then digested with restriction enzymes and the VNTR insert was cloned within the multiple cloning site of the reporter gene vectors either pGL3-promoter (pGL3P) or pGL3-basic (pGL3B) (Promega) in both orientations (forward and reverse) [restriction digests, vector maps and sequencing available at Marshall (2021 )]. In pGL3P the VNTR was located upstream of the SV40 minimal promoter to address action as an activator, and cloned in pGL3B to address putative function as an inherent transcriptional start site.