Gal4 dbd

To determine the subcellular localization of ThHSFA1 protein, the constructed vector 35S::ThHSFA1-GFP and control vector 35S::GFP were separately transformed into live onion epidermal cells using the biolistic bombardment method. After transformed and incubated on MS medium for 24 h at 22 °C in the dark, the transformed cells were visualized using confocal laser scanning microscopy (LSM700, Zeiss, Jena, Germany). To stain onion epidermal cell nuclei, 4′,6-diamidino-2-phenylindole (DAPI, 10 μg mL⁻¹) in phosphate-buffered saline was used.
For transcription activity analysis of ThHSFA1 protein, the full-length and partial cDNA of ThHSFA1 (encoding the N-terminus and C-terminus) were cloned into the yeast expression vector pGBKT7 and fused the GAL4 DBD (Clontech, Palo Alto, CA, USA). All constructs and the pGBKT7 vector (negative control) were introduced into Y2HGold yeast cells, which were cultured on SD/−Trp and SD/−Trp/X-a-Gal media for 2–3 days at 30 °C and then subjected to the β-galactosidase assay. The experiments were performed three times. All primers used are shown in Table S1.

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.