Journal: The Journal of biological chemistry

Article Title: Hepatitis C virus core protein binds to a DEAD box RNA helicase.

doi: 10.1074/jbc.274.22.15751

Figure Lengend Snippet: FIG. 1. Primary structures of DBX, PL10, and Ded1 and their interac- tions with HCV core protein in the yeast two-hybrid assay. A, alignment of deduced amino acid sequences of DBX (Gen- Banky accession number AF000982), PL10 (GenBanky accession number J04847), and Ded1p (GenBanky accession number X57278) is shown. Identical amino acids are shown as white on cyan. Conserved substi- tutions are shown as black on magenta. Dots represent gaps to optimize alignments, which were obtained using the Pileup pro- gram. B, two-hybrid assays showing interac- tion of HCV core protein with DBX and PL10 but not with Ded1p. Yeast strain Y190 was co-transformed with a plasmid express- ing the cytoplasmic domain of HCV fused to the GAL4 DNA binding domain and plas- mids expressing either a portion of DBX or the corresponding portions of PL10 or Ded1p fused to the GAL4 transcriptional activation domain. Transformants giving b-galactosid- ase activity (positive interactions) are blue. Control reactions of DBX, PL10, and Dep1p GAL 4 activation domain fusion proteins with GAL4 DNA binding domain alone were negative (data not shown).

Article Snippet: The amplified DNA was cloned into the GAL4 DNA binding domain fusion vector pAS2–1 (CLONTECH) to yield pAS2–1-HCV-core1–123.

Techniques: Y2H Assay, Transformation Assay, Plasmid Preparation, Binding Assay, Expressing, Activation Assay, Activity Assay, Control

Journal: Frontiers in Plant Science

Article Title: The Rice Abscisic Acid-Responsive RING Finger E3 Ligase OsRF1 Targets OsPP2C09 for Degradation and Confers Drought and Salinity Tolerance in Rice

doi: 10.3389/fpls.2021.797940

Figure Lengend Snippet: OsRF1 interacts with clade A OsPP2Cs. (A) Yeast two hybrid (Y2H) assay. AH109 yeast cells were co-transformed with the bait (pGBKT7-OsRF1) and prey (pGADT7-OsPP2Cs) constructs. Transformed cells were plated in color change selection media (SD-LT/X-α-Gal) or growth selection media (SD-LTH) and grown at 30°C for 3 days. (B) BiFC assay using pVYCE-OsRF1 and pVYNE-OsPP2Cs in rice protoplasts. The rice protoplast co-expressing OsRF1-VYCE and OsPP2C09-VYNE or OsPP2C50-VYNE in the presence or absence of MG132 was visualized using a confocal laser scanning microscope. YFP signals are represented in green, and chloroplast auto-fluorescence is represented in red. YFP; yellow fluorescent protein; BF, bright field. Bar = 10 μm.

Article Snippet: For yeast Y2H assay, full-length CDSs of synthetic OsRF1 was cloned into the pGBKT7 vector of Matchmaker Gal4 Two-Hybrid System 3 (Clontech, Palo Alto, CA, United States). pGADT7 vectors fused with nine OsPP2CAs (OsPP2C06, OsPP2C08, OsPP2C09, OsPP2C30, OsPP2C49, OsPP2C50, OsPP2C51, OsPP2C53, and OsPP2C68) were kindly provided by Dr. B-G. Kim (National Institute of Agricultural Sciences, South Korea).

Techniques: Y2H Assay, Transformation Assay, Construct, Selection, Bimolecular Fluorescence Complementation Assay, Expressing, Laser-Scanning Microscopy, Fluorescence

Journal: Molecular Plant Pathology

Article Title: Phytoplasma effector SWP1 induces witches’ broom symptom by destabilizing the TCP transcription factor BRANCHED1

doi: 10.1111/mpp.12733

Figure Lengend Snippet: SWP1 targets the shoot branching integrators BRC1 and BRC2. (a) Yeast two‐hybrid (Y2H) assay was used to examine the interaction of BD‐SWP1 with AD‐BRC1 and AD‐BRC2. Blue yeast colonies on SD/‐Trp‐Leu‐His‐Ade medium containing 40 μg/mL X‐α‐Gal (‐LWHA+X‐α‐Gal) indicate positive interaction between the two proteins. BD‐53/AD‐T, positive control; BD‐Lam/AD‐T, negative control; BD‐SWP1/AD, BD/AD‐BRC1 and BD/AD‐BRC2, vector controls. (b) Bimolecular fluorescence complementation (BiFC) assays confirm the interaction of SWP1 with BRC1 and BRC2 in Nicotiana benthamiana. Fluorescence signals were observed at 60 h after agro‐infiltration using a confocal laser scanning microscope. The plasmid combinations NYFP/CYFP, NYFP‐SWP1/CYFP, NYFP/CYFP‐BRC1 and NYFP/CYFP‐BRC2 were used as negative controls. Bars, 30 μm. (c, d) Y2H assays of SWP1 mutants (c) and BRC1 mutants (d). [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Interactions were determined by dropping 2 μL of yeast suspension (OD 600 = 0.3) onto SD/‐Trp‐Leu‐His‐Ade plates containing 40 μg/mL X‐α‐Gal (Clontech) (‐LWHA+X‐α‐Gal).

Techniques: Y2H Assay, Positive Control, Negative Control, Plasmid Preparation, Fluorescence, Laser-Scanning Microscopy

Journal:

Article Title: Interaction of the Arabidopsis E2F and DP Proteins Confers Their Concomitant Nuclear Translocation and Transactivation

doi: 10.1104/pp.010642

Figure Lengend Snippet: Activity for the interactions of AtE2Fs with DPa and DPb in the yeast two-hybrid assay. AtE2F1, AtE2F2, or AtE2F3 cDNA cloned in the pAD vector as prey was introduced into the yeast SFY526 with the DPa or DPb cDNA cloned in the pGBKT7 vector as bait. Galactosidase activity from the GAL4 UAS-LacZ reporter gene was measured. Data are shown as means ± sd (bars) of two independent experiments, which were carried out in triplicate.

Article Snippet: For yeast ( Saccharomyces cerevisiae ) vectors, fragments from the above GFP fusion constructs and p35S-AF3-F2C were cloned into the Eco RI or Nco I and Sal I sites of the pGBKT7 vector (CLONTECH, Palo Alto, CA) to generate pGB-AF1, -AF2, -AF3, -AF3-F2C, -DPa, and DPb.

Techniques: Activity Assay, Y2H Assay, Clone Assay, Plasmid Preparation

Journal:

Article Title: Interaction of the Arabidopsis E2F and DP Proteins Confers Their Concomitant Nuclear Translocation and Transactivation

doi: 10.1104/pp.010642

Figure Lengend Snippet: Analyses for the transactivation potencies of AtE2F proteins in yeast and the tobacco cells. A, Yeast assay. The indicated AtE2F cDNAs cloned in the GAL4 DNA binding domain expression vector pGBKT7 were introduced into the yeast SFY526, and β-galactosidase from the GAL4UAS-LacZ reporter gene was assayed. B, Transfection assay with tobacco cells. The cultured tobacco cells were cotransfected with the te2fR4-luc reporter construct and the indicated expression constructs of DPa and the wild-type AtE2F3 (AF3) or its derivatives with a deletion (AF3ΔC) or a replacement (AF3-F2C) of the C-terminal transactivation domain of AtE2F3. AF3C represents an expression plasmid for the C-terminal transactivation domain of AtE2F3 containing the SV40 NLS.

Article Snippet: For yeast ( Saccharomyces cerevisiae ) vectors, fragments from the above GFP fusion constructs and p35S-AF3-F2C were cloned into the Eco RI or Nco I and Sal I sites of the pGBKT7 vector (CLONTECH, Palo Alto, CA) to generate pGB-AF1, -AF2, -AF3, -AF3-F2C, -DPa, and DPb.

Techniques: Clone Assay, Binding Assay, Expressing, Plasmid Preparation, Transfection, Cell Culture, Construct