Pgbkt7 lam

The full-length TH1 coding region from the wild type and the mutants was amplified by PCR using primer BD-TH1F/R (see Supplementary Table S3) and cloned into the pGBKT7 vector (Clontech). The resultant constructs were co-transformed with the empty pGADT7 vector (Clontech) into yeast strain AH109 containing the GAL4-UAS-β-galactosidase reporter gene. The transformants were grown on the solid medium lacking leucine and tryptophan. The β-Galactosidase activity was detected using the Colony-lift Filter Assay according to the manufacturer’s user manual. For dimer formation assay, the full-length TH1 coding region from the wild type and the mutants was amplified by PCR using primer AD-TH1F/R (see Supplementary Table S3) and cloned into the pGADT7 vector. The resultant constructs were co-transformed with the pGBKT7 vector containing different TH1 alleles into yeast strain AH109. The transformants were grown on selective solid medium SD-Leu-Trp (DDO) or SD-Leu-Trp-His-Ade (QDO). Yeast colony co-transformed with pGBKT7-53 and pGADT7-T (Clontech) was used as positive control, while yeast colony co-transformed with pGBKT7-Lam and pGADT7-T (Clontech) was used as negative control.

Human MMP-9 gene was subcloned in pGBKT7, and DENV NS1, E, and NS4A were subcloned in pGADT7 respectively. control vectors pGBKT7, pGADT7, pGBKT7-p53, pGADT7-T, and pGBKT7-lam, and some reagents were purchased from Clontech Laboratories. All experimental procedures were done following the Matchmaker Gold Yeast 2-Hybrid System User Manual. Briefly, yeast strain AH109 cells were co-transformed with plasmid pGADT7 and plasmid pGBKT7. Transformed yeast cells were grown in SD-minus Trp/Leu plates (DDO) for 24–48 h, and then subcloned replica plated on SD-minus Trp/Leu/Ade/His plate (QDO) for another 48–96 h.

Saccharomyces cerevisiae strain AH109 and control vectors pGADT7, pGBKT7, pGADT7-T, pGBKT7-lam, and pGBKT7-p53 were purchased from Clontech (Mountain View, CA, USA). Yeast strain AH109 was co-transformed with the combination of the pGADT7 and the pGBKT7 plasmids. Transformed yeast cells containing both plasmids were first grown on SD-minus Trp/Leu plates (DDO) to maintain the two plasmids and then were subcloned replica plated on SD-minus Trp/Leu/Ade/His plate (QDO).

DF-1 cells and HEK 293 T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Plasmid pHSIE was kindly provided by Yi Zheng of the Shenzhen Key Lab of Gene and Antibody Therapy, Tsinghua University. Y2HGold yeast strain, Y187 yeast strain, pGADT7-Rec (cloning vector), pGBKT7 (cloning vector), pGADT7-T (control vector), pGBKT7-Lam (control vector), and pGBKT7-53 (control vector) were purchased from Clontech (Japan).

Saccharomyces cerevisiae strain AH109 and control vectors pGADT7, pGBKT7, pGADT7-T, pGBKT7-Lam, and pGBKT7-p53 were purchased from Clontech (Mountain View, CA, USA). Yeast strain AH109 was co-transformed with the combination of the pGADT7 and the pGBKT7 plasmids. Transformed yeast cells containing both plasmids were first grown on SD-minus Trp/Leu plates (DDO) to maintain the two plasmids and then were sub-cloned replica plated on SD-minus Trp/Leu/Ade/His plate (QDO).

Strain AH109 of Saccharomyces cerevisiae and vectors pGBKT7, pGADT7, pGBKT7-53, pGADT7-T, and pGBKT7-Lam were commercially provided by Clontech Laboratories Inc. (CA, USA) for protein-protein interaction analysis. For the construction of bait and prey vectors, the oligonucleotide primers, as shown in Supplementary Table S1, were used for gene amplification of MiglnB, MiargB, MiaccB1, and MiaccB2 lacking their signal peptide sequences. The amplicon carrying the mature MiPII-coding sequence was ligated into the EcoRI- and PstI-digested pGBKT7 to generate the bait vector pGBKT7-glnB. Similarly, MiargB, MiaccB1, and MiaccB2 lacking their signal peptide sequences were ligated into EcoRI- and XhoI-treated pGADT7 to generate the prey vectors, pGADT7-argB, pGADT7-accB1, and pGADT7-accB2, respectively. The plasmid pair between pGBKT7-glnB and pGADT7-argB, pGADT7-accB1, or pGADT7-accB2 was co-transformed into AH109 yeast separately by electroporation (Bio-Rad, USA), which were inoculated on SD-Trp-Leu plates according to the Clontech Yeast Protocols Handbook (Clontech, CA, USA). Expression of the lacZ reporter gene was determined by measuring β-galactosidase activity using 2-nitrophenyl-β-D-galactopyranoside as a substrate. The plasmid pairs pGBKT7-53 and pGADT7, and pGBKT7-Lam and pGADT7-T, served as a positive or negative control, respectively.