human kidney matchmakertm cdna library  (TaKaRa)

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Insights into the human cDNA: A descriptive study using library screening in yeast

doi: 10.1016/j.jgeb.2024.100427

Figure Lengend Snippet: Y2H cDNA library construction and screening procedure. A) The conventional procedure for cDNA library generation. B) Using the cDNA library in Y2H screens to identify protein–protein interactions. C) Using the cDNA library to screen for rescue genes involved in DNA repair. D) Screening randomly selected plasmids from the cDNA library to screen random samples from the library “Created with Biorender.com .”.

Article Snippet: The Human Kidney MatchmakerTM cDNA Library was purchased from Clontech (Cat. No 638816).

Techniques: cDNA Library Assay, Protein-Protein interactions

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Insights into the human cDNA: A descriptive study using library screening in yeast

doi: 10.1016/j.jgeb.2024.100427

Figure Lengend Snippet: The 5′ end truncated cDNAs identified, bp; base pairs.

Article Snippet: The Human Kidney MatchmakerTM cDNA Library was purchased from Clontech (Cat. No 638816).

Techniques: Variant Assay, Binding Assay, RNA Binding Assay

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Insights into the human cDNA: A descriptive study using library screening in yeast

doi: 10.1016/j.jgeb.2024.100427

Figure Lengend Snippet: mRNA priming in cDNA libraries. A) Normal priming through reverse transcriptase elongation of cDNA after oligo (dT) binding to true poly-A tails. B) Internal priming that leads to the generation of truncated transcripts due to internal A-rich regions and reverse transcriptase stalling. “Created with Biorender.com .”.

Article Snippet: The Human Kidney MatchmakerTM cDNA Library was purchased from Clontech (Cat. No 638816).

Techniques: Reverse Transcription, Binding Assay

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Insights into the human cDNA: A descriptive study using library screening in yeast

doi: 10.1016/j.jgeb.2024.100427

Figure Lengend Snippet: Mitochondrial hits status and their quantification.

Article Snippet: The Human Kidney MatchmakerTM cDNA Library was purchased from Clontech (Cat. No 638816).

Techniques:

Journal: PLoS ONE

Article Title: Direct Interaction of Selenoprotein R with Clusterin and Its Possible Role in Alzheimer’s Disease

doi: 10.1371/journal.pone.0066384

Figure Lengend Snippet: Plasmids carrying on the fetal brain cDNA library were co-transformed into the NpGBKT7- SelR′ -containing yeast and screened by the selection plate for the blue colonies (A). The interaction between SelR′ and Clu was verified by re-transformation of the plasmids NpGBKT7- SelR′ and pACT2- Clu into either AH109 (B) or Y2HGold (C) yeast cells. Yeast cells in B (1–3) were transformed with single NpGBKT7- SelR′ , NpGBKT7- SelR′ plus pACT2, and NpGBKT7- SelR′ plus pACT2- Clu plasmids, respectively. Yeast cells in C, D, E were transformed with NpGBKT7- SelR′ plus pACT2- Clu , pGBKT7- p53 plus pADT7- T (positive control), and pGBKT7- Lam plus pADT7- T (negative control), respectively, followed by the selection on SD/−Leu/−Trp/X-α-Gal/Aba plates.

Article Snippet: Matchmaker™ Gold yeast two-hybrid system, yeast strains Y2HGold and AH109, plasmids pACT2 and NpGBKT7, and human fetal brain cDNA library (using pACT2 as the vector) were purchased from Clontech Laboratories (Mountainview, CA, USA).

Techniques: cDNA Library Assay, Transformation Assay, Selection, Positive Control, Negative Control

Journal: PLoS ONE

Article Title: GRG5/AES Interacts with T-Cell Factor 4 (TCF4) and Downregulates Wnt Signaling in Human Cells and Zebrafish Embryos

doi: 10.1371/journal.pone.0067694

Figure Lengend Snippet: The AH109 yeast strain was co-transformed with the indicated constructs (central panel). Transformants were selected in medium lacking tryptophan and leucine (left panel) and activation of a Gal4-dependent ADE2 gene was examined by growth on selective plates additionally lacking adenine (right panel). Yeast cells containing both TCF4 and GRG5/AES grew in the selective media. pLAM and pACT2 plasmids were used as bait and prey negative controls respectively, while the β-catenin prey plasmid constitutes a positive control for the interaction assays.

Article Snippet: A commercial human fetal brain cDNA library in pACT2 (Clontech), was screened by PEG/LiAcetate co-transformation [ ] with the bait plasmid pAS2-TCF4 into the AH109 yeast reporter strain (which contains integrated copies of ADE2 and HIS3 reporter genes under control of Gal4-dependent promoters).

Techniques: Transformation Assay, Construct, Activation Assay, Plasmid Preparation, Positive Control

Journal: Virology Journal

Article Title: The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

doi: 10.1186/1743-422X-10-205

Figure Lengend Snippet: YFV NS5 (RNApol)- human eIF3L interaction in a yeast two - hybrid system. Yeast strain AH109 was co-transformed with pGBKT7-RNApol (or the empty or Lamin C human protein BD vector) and pACT2-eIF3L (or the empty or SV40 large T-antigen AD vector). The specific interaction was identified by the growth of transformants on SD medium (−LEU, -TRP, -HIS) and SD medium (−LEU, -TRP, -HIS, -ADE) with the activation of the reporter genes HIS 3 and ADE 2. C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

Article Snippet: The bait plasmid pGBKT7-RNApol and the pACT2 HeLa cell cDNA library (Clontech, USA) were sequentially introduced into the yeast cells using the EZ-Yeast Transformation kit - Single Vector Transformation protocol (Q.BIO gene, USA).

Techniques: Transformation Assay, Plasmid Preparation, Activation Assay, Negative Control, Positive Control

Journal: Virology Journal

Article Title: The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

doi: 10.1186/1743-422X-10-205

Figure Lengend Snippet: Determination of the YFV NS5 domain responsible for the interaction with eIF3L. The figure shows a representation of RNApol NS5 YFV and its fragments, with deletions (1–14) used to map the interaction domain with the eIF3L protein in the yeast two-hybrid system. The yeast was co-transformed with the pGBKT7-deletion constructs and pACT2-eIF3L. The interaction occurs between amino acids 368 and 448 (interaction domain, the region in black in the schematic representation), which was identified by the growth of transformants on SD medium (−LEU, -TRP, -HIS) and SD medium (−LEU, -TRP, -HIS, -ADE) with the activation of the reporter genes HIS 3 and ADE 2. C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

Article Snippet: The bait plasmid pGBKT7-RNApol and the pACT2 HeLa cell cDNA library (Clontech, USA) were sequentially introduced into the yeast cells using the EZ-Yeast Transformation kit - Single Vector Transformation protocol (Q.BIO gene, USA).

Techniques: Transformation Assay, Construct, Activation Assay, Negative Control, Positive Control

Journal: Virology Journal

Article Title: The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

doi: 10.1186/1743-422X-10-205

Figure Lengend Snippet: The eIF3L protein specifically interacts with the N - terminal region of the NS5 YFV interaction domain. The yeast was co-transformed with the pGBKT7 segments and pACT2-eIF3L (or the empty or large T-antigen SV40 AD vector). Segment 3 NS5 (ID) and eIF3L interaction was detected by activation of the reporter genes HIS 3 and ADE 2, which led to the growth of transformants on SD medium (−LEU, -TRP, -HIS) and SD medium (−LEU, -TRP, -HIS, -ADE). C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

Article Snippet: The bait plasmid pGBKT7-RNApol and the pACT2 HeLa cell cDNA library (Clontech, USA) were sequentially introduced into the yeast cells using the EZ-Yeast Transformation kit - Single Vector Transformation protocol (Q.BIO gene, USA).

Techniques: Transformation Assay, Plasmid Preparation, Activation Assay, Negative Control, Positive Control

Journal: Virology Journal

Article Title: The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

doi: 10.1186/1743-422X-10-205

Figure Lengend Snippet: The mapping of the terminal region residues of the interaction domain using the yeast two - hybrid system indicates a critical interaction with eIF3L. The yeast was co-transformed with the mutant pGBKT7 constructs and pACT2-eIF3L. A positive interaction is shown by the activation of the reporter gene HIS3 in the interaction of eIF3L with the mutants ID (D436N), ID (D436S), and ID (R439A/H442A), indicating that the mutations are not critical for the interaction. A negative interaction between mutant ID (F431A/W432A/V435A) and eIF3L is indicated by the absence of growth on plates lacking histidine (−His). C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

Article Snippet: The bait plasmid pGBKT7-RNApol and the pACT2 HeLa cell cDNA library (Clontech, USA) were sequentially introduced into the yeast cells using the EZ-Yeast Transformation kit - Single Vector Transformation protocol (Q.BIO gene, USA).

Techniques: Transformation Assay, Mutagenesis, Construct, Activation Assay, Negative Control, Positive Control

Journal: Virology Journal

Article Title: The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

doi: 10.1186/1743-422X-10-205

Figure Lengend Snippet: The interaction domains of other flaviviruses interact with eIF3L. The yeast cells were co-transformed with pGBKT7-ID ( Flavivirus members dengue virus types 3 and 4 and St. Louis encephalitis) and pACT2-eIF3L (or the empty or large T-antigen SV40 AD vector). The interaction domains of other flaviviruses (DENV types 3 and 4 and SLE) present a positive interaction with the eIF3L protein through the activation of the reporter genes HIS 3 and ADE 2. C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

Article Snippet: The bait plasmid pGBKT7-RNApol and the pACT2 HeLa cell cDNA library (Clontech, USA) were sequentially introduced into the yeast cells using the EZ-Yeast Transformation kit - Single Vector Transformation protocol (Q.BIO gene, USA).

Techniques: Transformation Assay, Plasmid Preparation, Activation Assay, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Cyclophilin B Interacts with Sodium-Potassium ATPase and Is Required for Pump Activity in Proximal Tubule Cells of the Kidney

doi: 10.1371/journal.pone.0013930

Figure Lengend Snippet: A: Yeast two hybrid assay. CDNA for CypB was cloned in frame into the yeast expression vector (pGBKT7) that harbours the GAL4 activation domain. The recombinant plasmids plus Human Kidney Matchmaker cDNA Library were co-transformed into AH109 yeast strain. The co-transformed cells were selected on Ade-/Leu-/His-/Trp-, 10 µM Aminotriazol dropout plates to monitor for growth. The positive control represents co-transformation of p53 and T-antigen in two-hybrid expression vectors, pGBKT7-P53 and pGADT7 (BD Biosciences, Clontech), respectively. B: GST-pull-down assays. CypB was fused in frame with the GST gene. GST and GST-HCypB products were immobilized on Sepharose 4B and incubated with COS-7 cells lysates expressing Na/K-β1 (pCMV-HA-Na/K). The bound proteins were analyzed by immunoblotting with anti-Na/K-β1 rabbit polyclonal antibody. C–E: Co-Immunoprecipitation assays. HK-2 lysates were immunoprecipitated with mouse monoclonal antibodies against Na/K-β1 or Na/K-α1 and rabbit polyclonal antibody against CypB. The immunoprecipitates were subjected to Western blotting analyses, as indicated in the figure. As control, ChromePure mouse IgG and ChromePure rabbit IgG were used. Figures 1B to 1E are representative of at least three independent experiments.

Article Snippet: A yeast two-hybrid screening was performed using a human kidney MATCHMAKER cDNA Library (BD Biosciences Clontech.

Techniques: Y2H Assay, Clone Assay, Expressing, Plasmid Preparation, Activation Assay, Recombinant, cDNA Library Assay, Transformation Assay, Positive Control, Incubation, Western Blot, Immunoprecipitation