The AD constructs used for Y3H were generated as described for the Y2H assays. To construct the pBridge-ULT1-DA1-C vector, the full-length coding sequence of ULT1 was cloned into the EcoRI and BamHI sites in the multiple cloning site I (MCS I) of the pBridge vector (Clontech) fused to the GAL4 DNA-binding domain, and the coding sequence of DA1-C was cloned into the MCS II site of the pBridge vector expressed as the “bridge” protein only in the absence of methionine. The pBridge-ULT1 vector was used as a control without the “bridge” protein. The constructs were co-transformed into the AH109 yeast strain. The presence of the transgenes was confirmed by growth on synthetic dextrose/−Leu/−Trp plates. The same OD-value co-transformed yeast cells were spread on plates containing synthetic dextrose/−Leu/−Trp/−His medium or synthetic dextrose/−Leu/−Trp/−His/−Met medium, with or without the expression of the third protein. Each transformed yeast was serially diluted tenfold (1×, 10×, and 100×) and spotted onto dropout synthetic medium.
Pbridge vector
Manufactured by Takara Bio
Sourced in United States
The PBridge vector is a plasmid used for cloning and expression of recombinant proteins in E. coli. It provides a platform for inserting target DNA sequences and facilitates their stable propagation and expression.
Automatically generated - may contain errors
Lab products found in correlation
Pgadt7 vector, by Takara Bio (3 mentions)
Matchmaker gal4 two hybrid system, by Takara Bio (2 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Platinum pfx dna polymerase, by Thermo Fisher Scientific (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Yeast handbook, by Takara Bio (1 mentions)
Ez yeast transformation kit, by MP Biomedicals (1 mentions)
Quikchange 2 xl, by Agilent Technologies (1 mentions)
Gal4 dna binding domain, by Takara Bio (1 mentions)
Ah109, by Takara Bio (1 mentions)
Matchmaker gold yeast two hybrid system, by Takara Bio (1 mentions)
Y2hgold yeast strain, by Takara Bio (1 mentions)
13 protocols using pbridge vector
1
Yeast Three-Hybrid Assay Protocol
2
Cloning and Expression of E2 Enzymes and MuRF1
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Using Superscript II and Platinum Pfx DNA polymerase (Invitrogen), we amplified by RT-PCR rat MuRF1, murine alpha-actin (a-actin), myosin heavy chain IIa (MHCIIa; MYH2 gene), the DNA fragment encoding amino acids 2–255 WT MHCIIa, UBE2E1 and UBE2L3 from either rat soleus muscles or murine C2C12 skeletal muscle cells. Then cDNAs encoding for E2 proteins and MuRF1 were cloned in the expression vectors pET28a (Novagen) and pGEX6P3, respectively, for the production of recombinant proteins in Escherichia coli (E. coli, BL21(DE3)). For mammalian in cellulo assays, UBE2L3 was sub-cloned in the expression vector pcDNA3.1 (Thermofisher), as described for flag-a-actin, MHCIIa-Flag, Myc-MuRF1 [45 (link)]. For yeast three-hybrid, UBE2 enzymes, MuRF3 and Large-T cDNAs were cloned in pGADT7 vector (containing the activation domain of GAL4) (Clontech). The pBridge vector (Clontech) allows the expression of two proteins: MuRF1 was cloned in the first Multiple Cloning Site (MCS I) in fusion with the binding domain of GAL4, and MHCIIa (residues 1–255) was cloned in the MCSII, leading to the pBridge::MuRF1/MHCIIa plasmid.
3
Yeast Two-Hybrid and Three-Hybrid Assays
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Full‐length ORFs of proteins shown in Table S3 were amplified from CS. The vectors for yeast two‐hybrid (Y2H) assays were generated using pGBKT7 (bait) and pGADT7 (prey). All the target genes were cloned into the pGBKT7 and pGADT7 vectors, respectively. The Y2H assay was carried out according to the manufacturer’s instructions of Matchmaker GAL4 Two‐Hybrid System (Clontech, USA). Briefly, specific bait and prey plasmids were co‐transformed into yeast strain AH109, and spread on selective medium SD/‐Trp/‐Leu (SD/‐TL). Transformed colonies were then dropped on selective medium SD/‐Trp/‐Leu/‐His/‐Ade (SD/‐TLHA) for protein‐protein interaction verification. Primers used here were listed in Table S3 .
For yeast three‐hybrid (Y3H) assay, the pBridge vector (Clontech, Palo Alto, California, USA) was used according to the manufacturer’s instructions. TaSTK1 was subcloned into the pBridge vector MCS I (multiple clone site) resulting in pBridge‐TaSTK1 and the TaAGL6 was subcloned into the pBridge‐TaSTK1 vector MCS II under the control of the Met‐repressible pMET25 promoter, resulting in pBridge‐TaSTK1‐proMET25‐TaAGL6 vector. The pBridge‐TaSTK1‐proMET25‐TaAGL6 and AD‐TaAG2 vectors were co‐transformed into AH109 cells. The colonies were streaked on the selection media without Trp, Leu, His, Ade and Met (SD/‐TLHAM) supplemented with or without Met.
For yeast three‐hybrid (Y3H) assay, the pBridge vector (Clontech, Palo Alto, California, USA) was used according to the manufacturer’s instructions. TaSTK1 was subcloned into the pBridge vector MCS I (multiple clone site) resulting in pBridge‐TaSTK1 and the TaAGL6 was subcloned into the pBridge‐TaSTK1 vector MCS II under the control of the Met‐repressible pMET25 promoter, resulting in pBridge‐TaSTK1‐proMET25‐TaAGL6 vector. The pBridge‐TaSTK1‐proMET25‐TaAGL6 and AD‐TaAG2 vectors were co‐transformed into AH109 cells. The colonies were streaked on the selection media without Trp, Leu, His, Ade and Met (SD/‐TLHAM) supplemented with or without Met.
4
Yeast Two-Hybrid Assays for Protein Interactions
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Yeast two-hybrid assays were performed using the Matchmaker GAL4 two-hybrid system (Clontech). The full and partial cDNAs of each gene were cloned into the pGADT7 and pGBKT7 vectors as prey and bait, respectively. The full and partial (RING, aa 1–104; CC, aa 121–213; WD40, aa 371–675) cDNAs of COP1 were cloned into the pGBK vector (as baits)23 (link). FKF1 was cloned into the pGAD vector (as prey) with full and partial cDNAs (LOV, aa 1–174; LOV+F-box, aa 1–283; F-box+KELCH, aa 174–618; KELCH, aa 283–618)11 (link). The clones were co-transformed into the yeast strain AH109. The pBridge vector (Clontech) was used for yeast three-hybrid assays. COP1 or SPA1 cDNAs were cloned into the multi-cloning site I of the pBridge vector, in which the binding domain BD-COP1 or BD-SPA1 fusion protein was expressed. Then, FKF1 was cloned into multi-cloning site II of the pBridge vector, in which FKF1 expression was controlled by the Met-repressible pMET25 promoter. These vectors were co-transformed into the yeast strain AH109. Yeast transformation was performed according to the Yeast Handbook (Clontech). The colonies were used for yeast cell growth assay, and a liquid assay using chlorophenol red-β-D-galactoside (CPRG) was used to measure β-galactosidase activity.
5
Yeast Three-Hybrid Screening for Protein-Protein Interactions
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Yeast three‐hybrid assays were performed using the MATCHMAKER GAL4 Two‐Hybrid System. The full‐length coding sequence of JAZ2 was cloned into the MCS I site of the pBridge vector (Clontech) and the coding sequence of wo was inserted into the MCS II site as the bridge protein. The full‐length coding sequence of SlMYC1 was cloned into the vector pGADT7. The vector pGADT7 and pBridge were co‐transformed into the yeast strain AH109. The transformed yeasts were incubated on the SD/‐Trp/‐Leu medium at 30 °C. After 3 days, the clones were transferred to the SD/‐Trp/‐Leu/‐Met medium and the transfer was repeated 3 times before being transferred to the SD/‐Ade/‐His/‐Leu/‐Trp medium for testing the interaction. Alternatively, the clones were incubated on the plates containing SD/‐Ade/‐His/‐Leu/‐Trp/‐Met to induce the wo expression. All plates were incubated at 30 °C for 4 days.
6
Yeast Two-Hybrid Analysis of Nef-Adaptor Interactions
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Y3H analysis was performed as previously described (Chaudhuri et al., 2007 (link), 2009 (link)). NL4-3 Nef or mouse tyrosinase cytosolic tail DNAs were subcloned into the pBridge vector (Clontech, CA) along with rat σ1 or σ2. Rat α and δ subunit DNAs were subcloned into the pGADT7 vector (Clonetech, CA). All the point mutants used in this study were generated by site-directed mutagenesis, using the QuikChange II XL (Agilent technologies, Santa Clara, CA). The canonical dileucine-containing tyrosinase tail construct was included as a positive control for the formation of a functional complex, and the σ1 subunit of AP-1 and the δ subunit of AP-3 were included as negative controls for self-activation. The mutations were verified by DNA sequencing. The Saccharomyces cerevisiae HF7c strain was cotransformed with the indicated pairs of pBridge and pGADT7 constructs, using EZ Yeast Transformation Kit (MP biomedicals, Solon, OH). Double transformants were selected and grown on plates lacking Leu, Trp, and Met (+HIS) for 3 days, then the colonies from each transformant were normalized and plated on + HIS plates and plates lacking Leu, Trp, Met, and HIS (−HIS) with/without 3-AT (3-amino-1,2,4-triazole) for 4 days.
7
Yeast Two-Hybrid Assay for Rice Protein Interactions
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The P7-2 and OSK1/OSK5/OSK20 were cloned into the pBridge vector (Clontech) to produce fusions with the GAL4 DNA-binding domain (BD) and Met promoter, respectively, and were transformed into yeast strain Y187. OsCUL1 was inserted into the pGADT7 vector (Clontech) to generate pGAD-OsCUL1 and was transformed into yeast strain AH109. Double transformants were selected on dropout meida (SD/-Met/-Leu/-Trp). Protein interactions were confirmed by growth on selective media (SD/-Met/-Leu/-Trp/-His/-Ade, X-α-gal). Serial dilution analysis was performed as described previously. S1 Table shows the primers used for construction of the aforementioned recombinant plasmids.
8
Construction and Validation of MdPP2AC-MdPTPA Protein Interaction
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Firstly, the CDS sequence of MdPTPA was ligated to the pBridge vector via single NotI site and then the CDS sequence of MdPP2AC was ligated into the pBridge vector (Clontech) using EcoRI and BamHI sites, constructing the recombination plasmids pBridge-MdPP2AC-MdPTPA. The full length of MdSnRK2.6 was ligated to pGADT7 vector via EcoRI and BamHI sites. Plasmids of pBridge-MdPP2AC-MdPTPA and pGADT7-MdSnRK2.6 were mated and transformed into the yeast strain AH109 (Clontech, USA), and the mating cultures were spread on stringent selective medium plates SD/-Leu/-Trp, SD/-Ade/-His/-Leu/-Trp, SD/-Ade/-His/-Leu/-Trp/-Met, respectively. The MdPTPA was cloned into MCS II of pBridge vector which was located downstream of an HA epitope and a second NLS, to form a bridge protein. The resulting fusion protein was conditionally expressed from the Met25 promoter in response to methionine levels in the medium (Appendix 3 Table S1
9
Yeast Two-Hybrid and Three-Hybrid Assay for Protein Interactions
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Y2H and Y3H were performed using the Matchmaker Gold Yeast Two-Hybrid System according to the manufacturer’s instructions (Clontech). For the Y2H, SDI1-BD clone was used as the bait, described previously (Aarabi et al., 2016 ), and mated with prey construct, MYC2-AD (Supplemental Table S5 ). For the Y3H Two combinations of constructs were generated in the pBridge vector (Clontech; Supplemental Table S5 ). SDI1BD-MYB28 expressed SDI1 fused to the DNA-BD (Gal4 DNA-binding domain) as well as MYB28 expressing as the bridging protein, without any attachment to the binding domain or activation domain. MYB28BD-SDI expressed MYB28 fused to the DNA-BD domain and SDI1 as the bridging protein. These two constructs were co-transformed with MYC2AD (Supplemental Table S5 ) as described previously (Aarabi et al., 2016 ). Positively transformed colonies were selected on the double, triple, and quadruple dropout plates as −Leu/−Met (DDO), −Leu/−Trp/−Met (TDO), and −His/−Leu/−Met/−Trp (QDO), respectively, either with or without X-α-Gal (X) and Aureobasidin.
10
Yeast Two-Hybrid Assay for Protein Interactions
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Y3H assays were performed as described previously64 (link). To construct pBridge-MED25-JAM1, MED25 CDS was cloned into the multiple cloning site (MCS) I of pBridge vector (Clontech) fused to the GAL4 BD domain, and JAM1 CDS was cloned into MCS II of the pBridge vector and expressed as the “bridge” protein only in the absence of Met. Constructs used for testing protein-protein interactions were co-transformed into yeast strain AH109. The presence of transgenes was confirmed by growing the yeast cells on the SD-L/T medium. Transformed yeast cells were spread on plates containing SD-L/T/A/H medium to assess the ERF1-MED25 interaction without the expression of JAM1 and on plates containing SD-L/T/A/H/M medium to induce JAM1 expression. Interactions were observed after 3 days of incubation at 28 °C. Primers used for plasmid construction are listed in Supplementary Table 2 .
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