Transactivation activity analysis was performed according to Hu et al. [24 (link)]. To investigate the transactivation activity of ThNAC4, the entire coding region and a series of truncated ThNAC4 genes were cloned into the pGBKT7 vector (Clontech, Palo Alto, CA, USA) and fused with a GAL4 DNA-binding domain. The yeast strain Y2H (Clontech, Palo Alto, CA, USA) was transformed with eight constructs and the pGBKT7 vector (negative control). The transformed strains were confirmed by sequencing and streaked on SD/-Trp/-His/X-α-gal plates. The trans-activation activities were evaluated based on their growth status. The plates were incubated for 3–5 days at 30 °C. Three independent experiments were performed.
Pgbkt7 vector
Manufactured by Takara Bio
Sourced in United States, Japan, Canada
The PGBKT7 vector is a plasmid used for gene expression in yeast. It contains a GAL1 promoter for inducible gene expression, a TRP1 selectable marker, and a 2-micron origin of replication for high-copy number maintenance in Saccharomyces cerevisiae.
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Lab products found in correlation
Pgadt7 vector, by Takara Bio (54 mentions)
Pgadt7, by Takara Bio (11 mentions)
Yeast strain ah109, by Takara Bio (5 mentions)
Matchmaker gold yeast two hybrid system, by Takara Bio (5 mentions)
Y2hgold, by Takara Bio (4 mentions)
X α gal, by Takara Bio (4 mentions)
Pgad gh vector, by Takara Bio (3 mentions)
Matchmaker gal4 two hybrid system, by Takara Bio (3 mentions)
Yeast protocols handbook, by Takara Bio (3 mentions)
Pgbkt7, by Takara Bio (3 mentions)
Pegfp n1 vector, by Takara Bio (2 mentions)
Y2h gold strain, by Takara Bio (2 mentions)
Pt3024 1, by Takara Bio (2 mentions)
Matchmaker system, by Takara Bio (2 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (2 mentions)
Matchmaker gal4 two hybrid system 3 kit, by Takara Bio (2 mentions)
Pcmv myc, by Takara Bio (2 mentions)
Pcmv ha, by Takara Bio (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Pcdna3.1 myc his vector, by Thermo Fisher Scientific (2 mentions)
151 protocols using pgbkt7 vector
1
Analyzing Transcriptional Activation of ThNAC4
2
Cloning and Characterization of Pear PLC and S-RNase Genes
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The diploid P. x bretschneideri Rehd. is the first pear species with its genome comprehensively sequenced (Wu et al., 2012b (link)). We compared the sequence of our cloned PLC cDNA fragments isolated from pollen tubes with that of the ‘DangshanSuli’ (P. x bretshneideri Rehd.) genome to determine the full-length PLC sequence. The full-length cDNA sequences of S7-RNase and S34-RNase (DQ414813) were based on the P. x bretschneideri Rehd. genome and GenBank sequences, respectively. The S7-RNase and S34-RNase cDNA sequences were separately cloned into the pGADT7 vector (Clontech). The PLC cDNA sequence was cloned into the pGBKT7 vector (Clontech). Comparing with the PLC gene mutation of the ‘Jinzhuili,’ we inserted 78 nucleotide residues, which encoded 26 amino acid residues, into the same mutant position in the ‘Dangshansuli’ PLC gene. The mutation of the ‘Dangshansuli’ PLC gene was also cloned into the pGBKT7 vector (Clontech). Each bait/prey pair was introduced to the AH109 yeast strain (Clontech). As a control for auto-activation false-positives, each bait was also co-transformed into the yeast strain with an empty AD vector, and each prey was co-transformed with an empty BD vector. The bait/prey pair colonies that grew on all selective media (Trp-Leu and Trp-Leu-Ade-His) were considered positive for interaction.
3
Yeast Two-Hybrid Screening of MdPP2AC, MdPTPA, and MdSnRK2.6
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The full length of MdPP2AC was ligated to the pGBKT7 vector (Clontech) as bait. The full length of MdPTPA and MdSnRK2.6 were ligated to pGADT7 vector as the prey, respectively. In addition, MdSnRK2.6 was ligated to the pGBKT7 vector (Clontech) as bait. The full length of MdPTPA was ligated to pGADT7 vector as the prey. Plasmids of the prey and bait were mated and transformed into the yeast strain Y2HGold (Clontech, USA), and the mating cultures were spread on stringent selective medium plates containing X-α-gal (20 mg mL-1) and 1 mg mL-1 Aureobasidin A (AbA). The plates were incubated at 30°C for 3 to 5 d and checked every 12 h for the development of blue color. Plasmids pGBKT7-Lam with pGADT7-T control vector and pGBKT7-MdPP2AC with pGADT7 were used as negative controls, and the pGBKT7-53 and pGADT7-T vectors were used as the positive controls. The primer sequences used are listed in Table S1
4
Yeast-based Transcriptional Activity Profiling of PeTCP Proteins
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The pGBKT7 vector (Clontech, Palo Alto, CA, United States) was used to study the transcriptional activity levels of four PeTCP proteins in yeast. The full-length PeTCPs open reading frames were PCR amplified and independently cloned into the pGBKT7 vector (Clontech; containing the GAL4 DNA-binding domain) using the gene-specific primers listed in Supplementary Table S5 . Subsequently, pGBKT7-PeTCP recombinant vectors, the positive control pGBKT7-53+pGADT7-T, and the negative control pGBKT7 empty plasmids were used to transform the yeast strain using the lithium acetate method. The transformed strains were further serially cultured on various SD selective media, including SD/-Trp and SD/-Trp/-His/-Ade/X-α-Gal, and incubated at 30°C for 3–5 days.
5
Yeast Two-Hybrid Plasmid Construction
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Two bait plasmids were constructed by cloning Met and GCE cDNAs with full-length open reading frames (ORFs) in the GAL4 DNA-binding domain of the pGBKT7 vector (Clontech, Mountain View, CA, USA). A prey plasmid was constructed by cloning the GAL4-AD fusion plasmid with a partial Taiman cDNA in the pGADT7 vector (Clontech), and using the following primers:
6
Yeast-based NF-Y Protein Interactions
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The coding sequences of NF-YA8, NF-YB1, NF-YC10, and NF-YC12 were amplified by PCR and subcloned into either the pGADT7 or pGBKT7 vector (Clontech). The prey and bait plasmids were verified by sequencing and subsequently transformed into yeast strain AH109. pGADT7-T was co-transformed with pGBKT7-53 as a positive control. The yeast cells were grown on SD lacking Leu and Trp (DDO) selection media at 30 °C for 3 d. Interactions were tested using SD/–Leu/–Trp/–His/–Ade (QDO) medium. QDO with X-α-Gal was used to detect the α-galactosidase activity of the yeast strains. Images were taken 5 d after the incubation.
In the yeast one-hybrid analysis, DNA fragments corresponding to the promoters of target genes were independently inserted into the pHIS2.0 plasmid (Clontech). NF-YC12 was fused to GAL4 transcriptional activation domain (AD). These constructs were transformed into the yeast strain AH109. A one-hybrid assay was performed following the manufacturer’s instructions (Clontech). Primers used for cloning are listed inSupplementary Table S1 .
In the yeast one-hybrid analysis, DNA fragments corresponding to the promoters of target genes were independently inserted into the pHIS2.0 plasmid (Clontech). NF-YC12 was fused to GAL4 transcriptional activation domain (AD). These constructs were transformed into the yeast strain AH109. A one-hybrid assay was performed following the manufacturer’s instructions (Clontech). Primers used for cloning are listed in
7
Exploring FXR1 and CMAS Interactions
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For two-hybrid screening, the full open reading frame of the human FXR1 (GenBank NM_005087.3) gene was subcloned into the pGBKT7 vector (28 (link)) (Clontech Laboratories, Inc., Mountainview, CA, USA) to construct pGBKT7-FXR1 (Table I ), which was used as the bait plasmid. In the co-immunoprecipitation assay, the full open reading frames of the human FXR1 gene and human CMP-N-acetyl-neuraminic acid synthetase (CMAS) gene (GenBank NM_018686.4) were subcloned into the pCMV-HA vector (28 (link)) (Clontech Laboratories, Inc.) and pCMV-Myc vector (28 (link)) (Clontech Laboratories, Inc.) to construct the recombinant vectors, pCMV-HA-FXR1 and pCMV-Myc-CMAS. In the colocalization assay, the full open reading frame of the human FXR1 gene and human CMAS gene were subcloned into the pEGFP-N1 vector (28 (link)) (Clontech Laboratories, Inc.) and pDsRed-Monomer-N1 vector (28 (link)) (Clontech Laboratories, Inc.), respectively, to construct pEGFP-N1-FXR1 and pDsRed-Monomer-N1-CMAS (Table I ). Additionally, the full open reading frame of the human FXR1 gene was inserted into the pcDNA3.1(−) vector (29 (link)) (Clontech Laboratories, Inc.) to generate the recombinant vector, pcDNA3.1(−)-FXR1 (Table I ), which was transfected into SH-SY5Y cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and biological effect of the interaction between FXR1P and CMAS was detected.
8
Cloning and Characterization of PHB2
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A full-length RSU1 cDNA was cloned from a human Kidney cDNA library (Clontech) by PCR using the following primers: 5ʹ-gcgaattcatgtccaagtctctgaagaagttggtg-3ʹ and 5ʹ-cggtcgacttatctgttcttggctgccaggggtttcc-3ʹ. The sequence of the RSU1 cDNA was confirmed by automated DNA sequencing. The RSU1 cDNA was inserted into the pGBKT7 vector (Clontech). The pGBKT7/RSU1 construct was used as bait to screen a human keratinocyte MATCHMAKER cDNA library following a previously described protocol (24 (link), 64 (link), 65 (link)). Six positive plasmids containing cDNA inserts with an identical size (1.1 kb) were selected and sequenced. They contain an identical cDNA fragment encoding the C-terminal fragment of PHB2 (residues 101–299). The full-length PHB2 cDNA was isolated from the human lung cDNA library by PCR using the following primers: 5ʹ-gctgaattcatggcccagaacttgaaggacttgg-3ʹ and 5ʹ-gagctcgagtcatttcttacccttgatgaggctg-3ʹ.
9
Yeast Two-Hybrid Screening for CDK5RAP2 Interactors
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CM1 domain of human CDK5RAP2 (58–196) [“(58–196)” indicates amino acids 58 to 196] was fused in-frame to the GAL4 DNA-binding domain using the pGBKT7 vector (CLONTECH). The resulting “bait” plasmid was used to screen a 9.5/10.5-day-old mouse embryo cDNA two-hybrid library (a gift from S. Hollenberg) in screens for CDK5RAP2-binding proteins by the yeast two-hybrid method as described previously [3 (link)].
10
Transcriptional Activation of BrERF72
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First, BrERF72 was inserted into the pGBKT7 vector (Clontech, USA) to fuse with GAL4 DNA-binding domain. Then, the fusion construct pGBKT7-BrERF72, positive control (pGBKT7-53 + pGADT7-T), and negative control (pGBKT7 vector) were transformed into yeast cells Gold Y2H using the lithium acetate method. The transcriptional activation ability of BrERF72 was evaluated based on the growth status and α-galactosidase activity of yeast cells that grow on SD medium without tryptophan (SD/-Trp), or tryptophan, histidine, and adenine (SD/-Trp-His-Ade).
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