Vp16 ad

For the MTH assay, the full-length open reading frame (ORF) of canine SGTA and REIC/Dkk-3 cDNA was cloned between the EcoRI and MluI sites of a pM GAL4 DNA-binding domain cloning plasmid (GAL4-DBD) and pVP16 transactivation domain cloning plasmid (VP16-AD) (Clontech Laboratories, Palo Alto, CA, USA), respectively. In addition, the full-length ORF of canine REIC/Dkk-3 cDNA was cloned between the XhoI and EcoRI sites of the pMACS KK HA(C) vector cloning plasmid (Miltenyi Biotec, Bergisch Gladbach, Germany). Primers were used in this study was described in Additional file 1: Table S1. Approximately 2 × 10⁵ 293 T cells per well in 24-well plates were co-transfected with 100 ng of pM, pVP16, pMACS KK HA(C), and pFR-Luc firefly luciferase reporter plasmid (Promega, Madison, WI, USA), and 0.2 ng of phRL-TK Renilla luciferase reporter plasmid (Promega). The cells were harvested 48 h after transfection, and the luciferase activity was measured using the dual-luciferase reporter assay system (Promega). The luciferase activity was normalized to the value of the Renilla luciferase activity [21 (link)].

For the mammalian cell two-hybrid assay, various lengths of human REIC/DKK-3 and SGTA cDNA were cloned into the BamHI and MluI sites of the pM GAL4 DNA-binding domain cloning plasmid (GAL4 DBD) and pVP16 transactivation domain cloning plasmid (VP16 AD) (Clontech Laboratories), respectively. The templates for each length of human REIC/DKK-3 and SGTA were generated by PCR amplification using appropriate primer pairs. Approximately 2 × 10⁵ 293T cells on 24-well plates were co-transfected with 100 ng of pM, 100 ng of pVP16, 100 ng of pFR-Luc firefly luciferase reporter plasmid (Promega) and 2 ng of phRL-TK Renilla luciferase reporter plasmid (Promega). The cells were harvested at 48 hours after transfection, and luciferase activity was measured using a dual-luciferase reporter assay system. Luciferase activity was normalized to Renilla luciferase activity [35 (link)]. DBD- and VP16-fused human SGTA constructs were introduced into 293T cells transfected with the REIC/DKK-3 constructs in the pMACS Kk.HA-C vector to examine the interference of SGTA-SGTA dimerization by REIC/DKK-3.