Structured Review

TaKaRa pgad gh
The matrix protein of VSV does not interact with PSMB6. (A) cDNAs encoding pre-LMP2 as well as pre-PSMB6 and mature PSMB6 were constructed in <t>pGAD</t> and used in <t>Y2H</t> assays against a pLEX VSV M construct. The empty pGAD (AD) or pLEX (LEX BD) vector was used as a negative control. The interaction was evaluated by quantification of β-galactosidase activity in liquid yeast cultures (see the Materials and Methods section). Bars represent standard deviations of the means from three independent experiments performed in triplicate. (B) Coimmunoprecipitation of M and PSMB6. N2A cells were transfected or not transfected (Non TF) for 24 h with plasmids carrying myc-pre-LMP2 (positive control), myc-pre-PSMB6, myc-mature-PSMB6, or myc-dystonin as a negative control and then infected with VSV (4 h; MOI, 3) before preparation of cell lysates. Following immunoprecipitation with an anti-M antibody or anti-myc antibody 9E10, cell extracts (inputs) and immune complexes (immunoprecipitates [IP]) were separated by SDS-PAGE and analyzed by Western blotting using anti-myc antibody 9E10 or M-specific antibody to reveal the presence of the different myc-LMP2, myc-PSMB6, and myc-dystonin constructs and M.
Pgad Gh, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pgad gh/product/TaKaRa
Average 91 stars, based on 11 article reviews
Price from $9.99 to $1999.99
pgad gh - by Bioz Stars, 2020-02
91/100 stars

Images

1) Product Images from "Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2"

Article Title: Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2

Journal: Journal of Virology

doi: 10.1128/JVI.01753-15

The matrix protein of VSV does not interact with PSMB6. (A) cDNAs encoding pre-LMP2 as well as pre-PSMB6 and mature PSMB6 were constructed in pGAD and used in Y2H assays against a pLEX VSV M construct. The empty pGAD (AD) or pLEX (LEX BD) vector was used as a negative control. The interaction was evaluated by quantification of β-galactosidase activity in liquid yeast cultures (see the Materials and Methods section). Bars represent standard deviations of the means from three independent experiments performed in triplicate. (B) Coimmunoprecipitation of M and PSMB6. N2A cells were transfected or not transfected (Non TF) for 24 h with plasmids carrying myc-pre-LMP2 (positive control), myc-pre-PSMB6, myc-mature-PSMB6, or myc-dystonin as a negative control and then infected with VSV (4 h; MOI, 3) before preparation of cell lysates. Following immunoprecipitation with an anti-M antibody or anti-myc antibody 9E10, cell extracts (inputs) and immune complexes (immunoprecipitates [IP]) were separated by SDS-PAGE and analyzed by Western blotting using anti-myc antibody 9E10 or M-specific antibody to reveal the presence of the different myc-LMP2, myc-PSMB6, and myc-dystonin constructs and M.
Figure Legend Snippet: The matrix protein of VSV does not interact with PSMB6. (A) cDNAs encoding pre-LMP2 as well as pre-PSMB6 and mature PSMB6 were constructed in pGAD and used in Y2H assays against a pLEX VSV M construct. The empty pGAD (AD) or pLEX (LEX BD) vector was used as a negative control. The interaction was evaluated by quantification of β-galactosidase activity in liquid yeast cultures (see the Materials and Methods section). Bars represent standard deviations of the means from three independent experiments performed in triplicate. (B) Coimmunoprecipitation of M and PSMB6. N2A cells were transfected or not transfected (Non TF) for 24 h with plasmids carrying myc-pre-LMP2 (positive control), myc-pre-PSMB6, myc-mature-PSMB6, or myc-dystonin as a negative control and then infected with VSV (4 h; MOI, 3) before preparation of cell lysates. Following immunoprecipitation with an anti-M antibody or anti-myc antibody 9E10, cell extracts (inputs) and immune complexes (immunoprecipitates [IP]) were separated by SDS-PAGE and analyzed by Western blotting using anti-myc antibody 9E10 or M-specific antibody to reveal the presence of the different myc-LMP2, myc-PSMB6, and myc-dystonin constructs and M.

Techniques Used: Construct, Plasmid Preparation, Negative Control, Activity Assay, Transfection, Positive Control, Infection, Immunoprecipitation, SDS Page, Western Blot

The matrix protein of VSV interacts with LMP2. (A) cDNAs encoding pre-LMP2, mature LMP2, and the 20-amino-acid N-terminal propeptide were constructed in pGAD and used in Y2H assays against a pLEX VSV M construct. The empty pGAD (AD) or pLEX (LEX BD) vector was used as a negative control. The interaction was evaluated by quantification of β-galactosidase activity in liquid yeast cultures as described in the Materials and Methods section. Bars represent standard deviations of the mean from three independent experiments performed in triplicate. *, P
Figure Legend Snippet: The matrix protein of VSV interacts with LMP2. (A) cDNAs encoding pre-LMP2, mature LMP2, and the 20-amino-acid N-terminal propeptide were constructed in pGAD and used in Y2H assays against a pLEX VSV M construct. The empty pGAD (AD) or pLEX (LEX BD) vector was used as a negative control. The interaction was evaluated by quantification of β-galactosidase activity in liquid yeast cultures as described in the Materials and Methods section. Bars represent standard deviations of the mean from three independent experiments performed in triplicate. *, P

Techniques Used: Construct, Plasmid Preparation, Negative Control, Activity Assay

2) Product Images from "Hrs recruits clathrin to early endosomes"

Article Title: Hrs recruits clathrin to early endosomes

Journal: The EMBO Journal

doi: 10.1093/emboj/20.17.5008

Fig. 3. The C-terminus of Hrs binds clathrin TD. ( A ). This illustrates the existence of a potential clathrin-binding motif within residues 770–775 of Hrs. Hrs is the only protein that has the clathrin box motif at the very C-terminus. (B–E) Interaction of Hrs with clathrin. ( B and C ) L40 reporter yeast cells were transformed with bait constructs in pLexA and prey constructs in pGAD. Reporter β-galactosidase activities (in arbitrary units) indicate binding and are represented as mean values of two independent experiments performed in duplicate. Error bars denote ± SEM. In (B), a clathrin triskelion (consisting of three heavy chains) is illustrated, with the terminal domain (TD), distal domain (DD) and hub domain (HD) indicated. ( D ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3) were immobilized on glutathione–Sepharose beads and incubated with pig brain cytosol. The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin with anti-clathrin heavy chain antibodies (upper panel). ( E ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3), were immobilized on glutathione–Sepharose beads and incubated with purified recombinant clathrin terminal domain (TD 1–579 ). The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin-TD 1–579 with anti-clathrin heavy chain (upper panel). Lane 4 represents the total amount of recombinant clathrin-TD 1–579 added to the beads.
Figure Legend Snippet: Fig. 3. The C-terminus of Hrs binds clathrin TD. ( A ). This illustrates the existence of a potential clathrin-binding motif within residues 770–775 of Hrs. Hrs is the only protein that has the clathrin box motif at the very C-terminus. (B–E) Interaction of Hrs with clathrin. ( B and C ) L40 reporter yeast cells were transformed with bait constructs in pLexA and prey constructs in pGAD. Reporter β-galactosidase activities (in arbitrary units) indicate binding and are represented as mean values of two independent experiments performed in duplicate. Error bars denote ± SEM. In (B), a clathrin triskelion (consisting of three heavy chains) is illustrated, with the terminal domain (TD), distal domain (DD) and hub domain (HD) indicated. ( D ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3) were immobilized on glutathione–Sepharose beads and incubated with pig brain cytosol. The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin with anti-clathrin heavy chain antibodies (upper panel). ( E ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3), were immobilized on glutathione–Sepharose beads and incubated with purified recombinant clathrin terminal domain (TD 1–579 ). The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin-TD 1–579 with anti-clathrin heavy chain (upper panel). Lane 4 represents the total amount of recombinant clathrin-TD 1–579 added to the beads.

Techniques Used: Binding Assay, Transformation Assay, Construct, Recombinant, Incubation, Centrifugation, SDS Page, Staining, Purification

3) Product Images from "The trans-Golgi network GRIP-domain proteins form ?-helical homodimers"

Article Title: The trans-Golgi network GRIP-domain proteins form ?-helical homodimers

Journal: Biochemical Journal

doi: 10.1042/BJ20041810

Dimerization of GCC88 constructs analysed by yeast two-hybrid interactions The indicated GCC88 constructs were generated as fusions with the DNA-binding protein LexA in pHybLex/Zeo and the GAL4 activation domain in pGAD GH. GCC88 refers to the full-length 775-amino-acid sequence, and the amino acid numbers denote truncated constructs. The pHybLex/Zeo and pGAD constructs were co-transformed into yeast reporter strain L40. Transformants were initially selected on medium lacking leucine and containing zeocin. The interaction between LexA and GAL4 fusions was assessed by HIS3 reporter gene activation as measured by the ability of transformants to grow on medium lacking histamine and by LacZ reporter gene activation using the liquid β-galactosidase assay. β-Galactosidase activity is given in Miller units. Assays were performed in triplicate.
Figure Legend Snippet: Dimerization of GCC88 constructs analysed by yeast two-hybrid interactions The indicated GCC88 constructs were generated as fusions with the DNA-binding protein LexA in pHybLex/Zeo and the GAL4 activation domain in pGAD GH. GCC88 refers to the full-length 775-amino-acid sequence, and the amino acid numbers denote truncated constructs. The pHybLex/Zeo and pGAD constructs were co-transformed into yeast reporter strain L40. Transformants were initially selected on medium lacking leucine and containing zeocin. The interaction between LexA and GAL4 fusions was assessed by HIS3 reporter gene activation as measured by the ability of transformants to grow on medium lacking histamine and by LacZ reporter gene activation using the liquid β-galactosidase assay. β-Galactosidase activity is given in Miller units. Assays were performed in triplicate.

Techniques Used: Construct, Generated, Binding Assay, Activation Assay, Sequencing, Transformation Assay, Activity Assay

Related Articles

Clone Assay:

Article Title: The Growth Factor Granulin Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription
Article Snippet: .. The plasmids pGBT9, pGAD GH, and pACT2 and the S. cerevisiae strain CG1945 for yeast two-hybrid system experiments were from Clontech Laboratories Inc. Truncated versions of cyclin T1 were cloned into the S. cerevisiae vector pGBT9, and cyclin T1 amino acids 1 to 708 [cyclin T1(1-708)] was also cloned into pGAD GH to confirm the interactions. .. Full-length CDK9 was cloned into pACT2.

Article Title: Z-DNA-binding proteins can act as potent effectors of gene expression invivo
Article Snippet: To construct a plasmid producing proteins without an AD, the Gal4 AD was deleted by Kpn I digestion from pGAD-GH (CLONTECH). .. Yeast vector pGNA was made by inserting a linker containing Nco I and Xho I sites at the Kpn I site, which allowed the cloning of desired protein genes in yeast.

Article Title: The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1
Article Snippet: The portion of the HIV-1BRU pol gene segment coding for the IN was cloned in frame with the DNA-binding LexA gene into the pBTM116 vector ( ). .. The cDNA of wild-type (WT) EED ( ) was inserted into the Eco RI and Not I sites of the Gal4 transcription activation domain vector pGAD3S2X, a modified version of the pGAD GH (Clontech) containing a Not I site in its polylinker.

Article Title: Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair
Article Snippet: .. RAD6 was cloned into pAS2-1 and a derivative of pGAD GH (Clontech). .. Truncations of RAD18 and RAD5 were constructed using internal restriction sites.

Article Title: Hrs recruits clathrin to early endosomes
Article Snippet: .. For use in the two-hybrid system, constructs were cloned into pLexA/pBTM116 ( ) as bait and pGAD GH (Clontech) as prey. .. For expression of GST fusion proteins in Escherichia coli , pGEX-Hrs or the deletion constructs indicated were obtained by subcloning the respective cDNAs into the polylinker sites of pGEX-6P (Amersham Pharmacia). pGEX-2T-clathrin1–579 was a gift from Dr Jim Keen ( ).

Amplification:

Article Title: Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair
Article Snippet: Plasmid mms2 Δ ::HIS3 was constructed by amplifying 511 bp of the upstream and 420 bp of the downstream flanking regions of MMS2 by PCR from genomic DNA and combining the fragments with a HIS3 cassette in pBluescript-SK(+) (Stratagene). rad18 Δ ::TRP1 contains 464 bp of the RAD18 upstream flanking region and the C-terminal part of the open reading frame (ORF; 520 bp) interrupted by the TRP1 locus in pBluescript-SK(+). rad30 Δ ::HIS 3 was constructed by inserting a 3076 bp PCR fragment containing the RAD30 gene with flanking regions into pBluescript-SK(+) and replacing the Stu I– Ehe I fragment, containing the entire ORF, with the HIS3 marker. rad5 Δ ::HIS3 was constructed similarly by amplification of the RAD5 ORF, insertion into pBluescript-SK(+) and replacement of the internal Bcl I– Bgl II fragment with HIS3 . .. RAD6 was cloned into pAS2-1 and a derivative of pGAD GH (Clontech).

Construct:

Article Title: Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2
Article Snippet: .. To obtain the plasmid constructs used in the yeast two-hybrid (Y2H) assay, cDNAs encoding premature or mature LMP2 (obtained from the Y2H screen) and PSMB6 (synthetic gene obtained from Biovalley) were constructed in pGAD-GH (Clontech), a derivative of pGAD-GE. cDNAs coding for VSV M (serotype Indiana, strain Orsay) as well as all VSV M mutants were constructed in LexA (Clontech) ( ). ..

Article Title: The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1
Article Snippet: The sequence of the construct was verified by DNA sequencing, and the expression of recombinant fusion protein in yeast was confirmed by gel electrophoresis and immunoblot analysis with anti-IN polyclonal antibody as described below. (ii) Generation of the pGAD-EED (Fig. ). .. The cDNA of wild-type (WT) EED ( ) was inserted into the Eco RI and Not I sites of the Gal4 transcription activation domain vector pGAD3S2X, a modified version of the pGAD GH (Clontech) containing a Not I site in its polylinker.

Article Title: The 14-kDa Dynein Light Chain-Family Protein Dlc1 Is Required for Regular Oscillatory Nuclear Movement and Efficient Recombination during Meiotic Prophase in Fission Yeast
Article Snippet: .. A bait plasmid containing a segment of Kms1 protein (amino acid residues 202–607) fused to the Gal4 DNA-binding domain (Gal4BD) in pAS2.1 was used to screen an S. pombe cDNA library constructed with pGAD GH ( CLONTECH ). .. Various parts of Kms1 protein as well as Dlc1 were fused to the activation domain on pACT2 by using PCR-generated fragments.

Article Title: The trans-Golgi network GRIP-domain proteins form ?-helical homodimers
Article Snippet: pHybLex-GCC88, pHybLex-GCC88aa1-330 , pHybLex-GCC88aa331-775 , pGAD-GCC88 and pGAD-GCC88aa441-775 were obtained by subcloning the full-length [ ] and partial GCC88 cDNAs, isolated from full-length GCC88 cDNA by digestion with restriction enzymes, into the polylinker sites of pHybLex/Zeo (LexA-binding domain; Invitrogen) and pGAD GH (GAL4 activation domain; Clontech, Palo Alto, CA, U.S.A.) respectively. .. The pHybLex/Zeo and pGAD GH constructs were co-transformed in to the Saccharomyces cerevisiae reporter stain L40 [ ] using a lithium acetate method [ ].

Article Title: Rdp1, a Novel Zinc Finger Protein, Regulates the DNA Damage Response of rhp51+ from Schizosaccharomyces pombe
Article Snippet: The DRErhp51+ -lacZ reporter plasmid pRW3-F3 was constructed by inserting a 150-bp Eco RI fragment of pHis33-F3 containing three copies of the DREs immediately upstream of lacZ in the CEN-ARS- TRP1 plasmid pRW95-3 ( ). .. The S. pombe cDNA expression library based on a 2μm- LEU2 plasmid, pGAD GH, was purchased from Clontech (catalog no. XL4000AA).

Article Title: Z-DNA-binding proteins can act as potent effectors of gene expression invivo
Article Snippet: .. Reporter vectors were constructed by using the pLacZi vector as a template and expression vectors were constructed by using either pACT2 or pGAD-GH (CLONTECH). ..

Article Title: Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair
Article Snippet: Plasmid mms2 Δ ::HIS3 was constructed by amplifying 511 bp of the upstream and 420 bp of the downstream flanking regions of MMS2 by PCR from genomic DNA and combining the fragments with a HIS3 cassette in pBluescript-SK(+) (Stratagene). rad18 Δ ::TRP1 contains 464 bp of the RAD18 upstream flanking region and the C-terminal part of the open reading frame (ORF; 520 bp) interrupted by the TRP1 locus in pBluescript-SK(+). rad30 Δ ::HIS 3 was constructed by inserting a 3076 bp PCR fragment containing the RAD30 gene with flanking regions into pBluescript-SK(+) and replacing the Stu I– Ehe I fragment, containing the entire ORF, with the HIS3 marker. rad5 Δ ::HIS3 was constructed similarly by amplification of the RAD5 ORF, insertion into pBluescript-SK(+) and replacement of the internal Bcl I– Bgl II fragment with HIS3 . .. RAD6 was cloned into pAS2-1 and a derivative of pGAD GH (Clontech).

Article Title: MCAF Mediates MBD1-Dependent Transcriptional Repression
Article Snippet: .. Yeast strain CG-1945 carrying pAS2-1-TRD of MBD1 (amino acids 529 to 592 [isoform v1] or 473 to 536 [isoform v3]) ( ) was transformed with the HeLa cDNA libraries constructed in pGAD-GH (Clontech). ..

Article Title: Zyxin, a Regulator of Actin Filament Assembly, Targets the Mitotic Apparatus by Interacting with H-Warts/Lats1 Tumor Suppressor
Article Snippet: .. An L40 strain carrying pBTM116HA/h-warts (amino acids 394–675) was transformed with the HeLa cDNA library constructed in pGAD-GH (Clontech) by electroporation. ..

Article Title: Hrs recruits clathrin to early endosomes
Article Snippet: .. For use in the two-hybrid system, constructs were cloned into pLexA/pBTM116 ( ) as bait and pGAD GH (Clontech) as prey. .. For expression of GST fusion proteins in Escherichia coli , pGEX-Hrs or the deletion constructs indicated were obtained by subcloning the respective cDNAs into the polylinker sites of pGEX-6P (Amersham Pharmacia). pGEX-2T-clathrin1–579 was a gift from Dr Jim Keen ( ).

Expressing:

Article Title: The Growth Factor Granulin Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription
Article Snippet: The plasmids pGBT9, pGAD GH, and pACT2 and the S. cerevisiae strain CG1945 for yeast two-hybrid system experiments were from Clontech Laboratories Inc. Truncated versions of cyclin T1 were cloned into the S. cerevisiae vector pGBT9, and cyclin T1 amino acids 1 to 708 [cyclin T1(1-708)] was also cloned into pGAD GH to confirm the interactions. .. Plasmids for the expression of glutathione S -transferase (GST) fusion proteins were the full-length cyclin T1 [T1(1-726)] from K. A. Jones ( ) and the C-terminal truncations T1(1-250), T1(1-300), T1(1-479), and T1(1-555) from B. M. Peterlin ( ).

Article Title: Z-DNA-binding proteins can act as potent effectors of gene expression invivo
Article Snippet: Paragraph title: Construction of Yeast Expression Vectors for Z-DNA-Binding Proteins With or Without AD Fusion. ... To construct a plasmid producing proteins without an AD, the Gal4 AD was deleted by Kpn I digestion from pGAD-GH (CLONTECH).

Article Title: The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1
Article Snippet: The sequence of the construct was verified by DNA sequencing, and the expression of recombinant fusion protein in yeast was confirmed by gel electrophoresis and immunoblot analysis with anti-IN polyclonal antibody as described below. (ii) Generation of the pGAD-EED (Fig. ). .. The cDNA of wild-type (WT) EED ( ) was inserted into the Eco RI and Not I sites of the Gal4 transcription activation domain vector pGAD3S2X, a modified version of the pGAD GH (Clontech) containing a Not I site in its polylinker.

Article Title: Rdp1, a Novel Zinc Finger Protein, Regulates the DNA Damage Response of rhp51+ from Schizosaccharomyces pombe
Article Snippet: .. The S. pombe cDNA expression library based on a 2μm- LEU2 plasmid, pGAD GH, was purchased from Clontech (catalog no. XL4000AA). .. The truncated cDNA sequence of rdp1 + was generated by PCR using oligonucleotides OR1F-Bam (5′-CACGGGATCCAACACTCCCACCGTAG-3′) and OR540R-EcoR (5′-GGGGTTGGAATCAGGCACTTGAC-3′) as primers and pGAD236 as the template.

Article Title: Z-DNA-binding proteins can act as potent effectors of gene expression invivo
Article Snippet: .. Reporter vectors were constructed by using the pLacZi vector as a template and expression vectors were constructed by using either pACT2 or pGAD-GH (CLONTECH). ..

Article Title: A Chimeric Protein Containing the N Terminus of the Adeno-Associated Virus Rep Protein Recognizes Its Target Site in an In Vivo Assay
Article Snippet: .. An Aat II- Hin dIII fragment of pGAD GH (Clontech) containing the full-length promoter was subcloned into the pG expression plasmid series to replace a corresponding fragment containing a truncated version of the ADH1 promoter, giving rise to pADH.Rep68, pADH.Rep78, pADH.Rep.AD, pADH.Rep.LZ.AD, pADH.Rep.TZ.AD, and pADH.Rep.TZ. .. Plasmid pADH.Keratin was kindly provided by Jeanette Ducut.

Article Title: Hrs recruits clathrin to early endosomes
Article Snippet: For expression in mammalian cells with the T7 RNA polymerase vaccinia virus system, constructs were cloned behind the myc epitope of pGEM-myc4 ( ). .. For use in the two-hybrid system, constructs were cloned into pLexA/pBTM116 ( ) as bait and pGAD GH (Clontech) as prey.

Modification:

Article Title: The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1
Article Snippet: .. The cDNA of wild-type (WT) EED ( ) was inserted into the Eco RI and Not I sites of the Gal4 transcription activation domain vector pGAD3S2X, a modified version of the pGAD GH (Clontech) containing a Not I site in its polylinker. .. This resulted in a Gal4 activation domain AD-EED hybrid.

Article Title: Z-DNA-binding proteins can act as potent effectors of gene expression invivo
Article Snippet: Vector constructs were modified from the vectors used in the MATCHMAKER yeast hybrid system (CLONTECH). .. Reporter vectors were constructed by using the pLacZi vector as a template and expression vectors were constructed by using either pACT2 or pGAD-GH (CLONTECH).

Transformation Assay:

Article Title: MCAF Mediates MBD1-Dependent Transcriptional Repression
Article Snippet: .. Yeast strain CG-1945 carrying pAS2-1-TRD of MBD1 (amino acids 529 to 592 [isoform v1] or 473 to 536 [isoform v3]) ( ) was transformed with the HeLa cDNA libraries constructed in pGAD-GH (Clontech). ..

Article Title: Live-Cell Imaging Reveals Multiple Interactions between Epstein-Barr Virus Nuclear Antigen 1 and Cellular Chromatin during Interphase and Mitosis
Article Snippet: A premade HeLa cell random cDNA library fused with GAL4 AD in pGAD-GH (Clontech) was used. .. The transformed yeasts were selected on agar plates with a double (−Leu/−Trp) or a triple (−Leu/−Trp/−His) dropout of the essential amino acids.

Article Title: Centrosomal Proteins CG-NAP and Kendrin Provide Microtubule Nucleation Sites by Anchoring ?-Tubulin Ring Complex
Article Snippet: .. The resultant plasmid was transformed to the yeast reporter strain AH109 together with a HeLa cDNA library fused to the Gal4 activation domain (Gal4ad) in pGAD GH ( Clontech ). .. Bacterial expression plasmids for GST-tagged CG-NAP3510–3829 and His6 -tagged calmodulin 2 were constructed by inserting the corresponding cDNA fragments into pGEX4T and pRSET (Invitrogen), respectively.

Article Title: Zyxin, a Regulator of Actin Filament Assembly, Targets the Mitotic Apparatus by Interacting with H-Warts/Lats1 Tumor Suppressor
Article Snippet: .. An L40 strain carrying pBTM116HA/h-warts (amino acids 394–675) was transformed with the HeLa cDNA library constructed in pGAD-GH (Clontech) by electroporation. ..

Over Expression:

Article Title: Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair
Article Snippet: RAD6 was cloned into pAS2-1 and a derivative of pGAD GH (Clontech). .. The constructs for VSV RAD5 overexpression are based on the integrative plasmid YIplac211 carrying the URA3 marker , the GAL1-10 promoter, a VSV epitope and a transcription terminator derived from pGAD424.

Derivative Assay:

Article Title: Rdp1, a Novel Zinc Finger Protein, Regulates the DNA Damage Response of rhp51+ from Schizosaccharomyces pombe
Article Snippet: The S. pombe cDNA expression library based on a 2μm- LEU2 plasmid, pGAD GH, was purchased from Clontech (catalog no. XL4000AA). .. To disrupt the rdp1 + gene, a 5.3-kb Eco RI- Xho I fragment containing the entire gene was derived from the cosmid SPAC1B1 (a gift from Rhian Gwilliam at The Sanger Centre) and subcloned into pBSIIKS(+) to create plasmid prdp1-830.

Article Title: Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair
Article Snippet: RAD6 was cloned into pAS2-1 and a derivative of pGAD GH (Clontech). .. The constructs for VSV RAD5 overexpression are based on the integrative plasmid YIplac211 carrying the URA3 marker , the GAL1-10 promoter, a VSV epitope and a transcription terminator derived from pGAD424.

Electroporation:

Article Title: Zyxin, a Regulator of Actin Filament Assembly, Targets the Mitotic Apparatus by Interacting with H-Warts/Lats1 Tumor Suppressor
Article Snippet: .. An L40 strain carrying pBTM116HA/h-warts (amino acids 394–675) was transformed with the HeLa cDNA library constructed in pGAD-GH (Clontech) by electroporation. ..

Generated:

Article Title: Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2
Article Snippet: To obtain the plasmid constructs used in the yeast two-hybrid (Y2H) assay, cDNAs encoding premature or mature LMP2 (obtained from the Y2H screen) and PSMB6 (synthetic gene obtained from Biovalley) were constructed in pGAD-GH (Clontech), a derivative of pGAD-GE. cDNAs coding for VSV M (serotype Indiana, strain Orsay) as well as all VSV M mutants were constructed in LexA (Clontech) ( ). .. Mutants were generated in pLEX for M and in pGAD for LMP2.

Article Title: Rdp1, a Novel Zinc Finger Protein, Regulates the DNA Damage Response of rhp51+ from Schizosaccharomyces pombe
Article Snippet: The S. pombe cDNA expression library based on a 2μm- LEU2 plasmid, pGAD GH, was purchased from Clontech (catalog no. XL4000AA). .. The truncated cDNA sequence of rdp1 + was generated by PCR using oligonucleotides OR1F-Bam (5′-CACGGGATCCAACACTCCCACCGTAG-3′) and OR540R-EcoR (5′-GGGGTTGGAATCAGGCACTTGAC-3′) as primers and pGAD236 as the template.

Article Title: Hrs recruits clathrin to early endosomes
Article Snippet: The Hrs constructs indicated were generated by PCR with mouse Hrs ( ) as the template. .. For use in the two-hybrid system, constructs were cloned into pLexA/pBTM116 ( ) as bait and pGAD GH (Clontech) as prey.

DNA Sequencing:

Article Title: The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1
Article Snippet: The sequence of the construct was verified by DNA sequencing, and the expression of recombinant fusion protein in yeast was confirmed by gel electrophoresis and immunoblot analysis with anti-IN polyclonal antibody as described below. (ii) Generation of the pGAD-EED (Fig. ). .. The cDNA of wild-type (WT) EED ( ) was inserted into the Eco RI and Not I sites of the Gal4 transcription activation domain vector pGAD3S2X, a modified version of the pGAD GH (Clontech) containing a Not I site in its polylinker.

Y2H Assay:

Article Title: Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2
Article Snippet: .. To obtain the plasmid constructs used in the yeast two-hybrid (Y2H) assay, cDNAs encoding premature or mature LMP2 (obtained from the Y2H screen) and PSMB6 (synthetic gene obtained from Biovalley) were constructed in pGAD-GH (Clontech), a derivative of pGAD-GE. cDNAs coding for VSV M (serotype Indiana, strain Orsay) as well as all VSV M mutants were constructed in LexA (Clontech) ( ). ..

Sequencing:

Article Title: The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1
Article Snippet: The sequence of the construct was verified by DNA sequencing, and the expression of recombinant fusion protein in yeast was confirmed by gel electrophoresis and immunoblot analysis with anti-IN polyclonal antibody as described below. (ii) Generation of the pGAD-EED (Fig. ). .. The cDNA of wild-type (WT) EED ( ) was inserted into the Eco RI and Not I sites of the Gal4 transcription activation domain vector pGAD3S2X, a modified version of the pGAD GH (Clontech) containing a Not I site in its polylinker.

Article Title: Rdp1, a Novel Zinc Finger Protein, Regulates the DNA Damage Response of rhp51+ from Schizosaccharomyces pombe
Article Snippet: The DNA structures of all plasmids were confirmed by restriction analysis and in some cases by sequencing. .. The S. pombe cDNA expression library based on a 2μm- LEU2 plasmid, pGAD GH, was purchased from Clontech (catalog no. XL4000AA).

Article Title: Zyxin, a Regulator of Actin Filament Assembly, Targets the Mitotic Apparatus by Interacting with H-Warts/Lats1 Tumor Suppressor
Article Snippet: An L40 strain carrying pBTM116HA/h-warts (amino acids 394–675) was transformed with the HeLa cDNA library constructed in pGAD-GH (Clontech) by electroporation. .. Plasmids harboring cDNA were recovered from positive colonies and the nucleotide sequence of plasmid DNA which conferred the LacZ+ phenotype on L40 containing pBTM116HA/h-warts (396–657) were determined.

Binding Assay:

Article Title: Centrosomal Proteins CG-NAP and Kendrin Provide Microtubule Nucleation Sites by Anchoring ?-Tubulin Ring Complex
Article Snippet: The cDNA fragment encoding CG-NAP3510–3828 was fused to the Gal4 DNA binding domain (Gal4bd) by subcloning into pGCKT7 ( Clontech ). .. The resultant plasmid was transformed to the yeast reporter strain AH109 together with a HeLa cDNA library fused to the Gal4 activation domain (Gal4ad) in pGAD GH ( Clontech ).

Nucleic Acid Electrophoresis:

Article Title: The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1
Article Snippet: The sequence of the construct was verified by DNA sequencing, and the expression of recombinant fusion protein in yeast was confirmed by gel electrophoresis and immunoblot analysis with anti-IN polyclonal antibody as described below. (ii) Generation of the pGAD-EED (Fig. ). .. The cDNA of wild-type (WT) EED ( ) was inserted into the Eco RI and Not I sites of the Gal4 transcription activation domain vector pGAD3S2X, a modified version of the pGAD GH (Clontech) containing a Not I site in its polylinker.

Mutagenesis:

Article Title: Z-DNA-binding proteins can act as potent effectors of gene expression invivo
Article Snippet: The engineered Zα motif, hZaaADAR , and the mutant, hZa′bADAR (I172F and N173A in hZabADAR ), have been described ( , ). .. To construct a plasmid producing proteins without an AD, the Gal4 AD was deleted by Kpn I digestion from pGAD-GH (CLONTECH).

Article Title: The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1
Article Snippet: The cDNA of wild-type (WT) EED ( ) was inserted into the Eco RI and Not I sites of the Gal4 transcription activation domain vector pGAD3S2X, a modified version of the pGAD GH (Clontech) containing a Not I site in its polylinker. .. In the hybrid mutant AD-EED-305A4 the tetrapeptide motif HNRY from positions 305 to 308 was replaced by the tetrapeptide AAAA, and in AD-EED-300AI the dipeptide motif ST at positions 300 and 301 was replaced by AI. (iii) The two-hybrid assays were performed as described in a previous study ( ).

Article Title: Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair
Article Snippet: RAD6 was cloned into pAS2-1 and a derivative of pGAD GH (Clontech). .. The point mutation in the RING finger of RAD5 was created by PCR mutagenesis.

Isolation:

Article Title: The trans-Golgi network GRIP-domain proteins form ?-helical homodimers
Article Snippet: .. pHybLex-GCC88, pHybLex-GCC88aa1-330 , pHybLex-GCC88aa331-775 , pGAD-GCC88 and pGAD-GCC88aa441-775 were obtained by subcloning the full-length [ ] and partial GCC88 cDNAs, isolated from full-length GCC88 cDNA by digestion with restriction enzymes, into the polylinker sites of pHybLex/Zeo (LexA-binding domain; Invitrogen) and pGAD GH (GAL4 activation domain; Clontech, Palo Alto, CA, U.S.A.) respectively. .. The pHybLex/Zeo and pGAD GH constructs were co-transformed in to the Saccharomyces cerevisiae reporter stain L40 [ ] using a lithium acetate method [ ].

Subcloning:

Article Title: The trans-Golgi network GRIP-domain proteins form ?-helical homodimers
Article Snippet: .. pHybLex-GCC88, pHybLex-GCC88aa1-330 , pHybLex-GCC88aa331-775 , pGAD-GCC88 and pGAD-GCC88aa441-775 were obtained by subcloning the full-length [ ] and partial GCC88 cDNAs, isolated from full-length GCC88 cDNA by digestion with restriction enzymes, into the polylinker sites of pHybLex/Zeo (LexA-binding domain; Invitrogen) and pGAD GH (GAL4 activation domain; Clontech, Palo Alto, CA, U.S.A.) respectively. .. The pHybLex/Zeo and pGAD GH constructs were co-transformed in to the Saccharomyces cerevisiae reporter stain L40 [ ] using a lithium acetate method [ ].

Article Title: Centrosomal Proteins CG-NAP and Kendrin Provide Microtubule Nucleation Sites by Anchoring ?-Tubulin Ring Complex
Article Snippet: The cDNA fragment encoding CG-NAP3510–3828 was fused to the Gal4 DNA binding domain (Gal4bd) by subcloning into pGCKT7 ( Clontech ). .. The resultant plasmid was transformed to the yeast reporter strain AH109 together with a HeLa cDNA library fused to the Gal4 activation domain (Gal4ad) in pGAD GH ( Clontech ).

Article Title: Hrs recruits clathrin to early endosomes
Article Snippet: For use in the two-hybrid system, constructs were cloned into pLexA/pBTM116 ( ) as bait and pGAD GH (Clontech) as prey. .. For expression of GST fusion proteins in Escherichia coli , pGEX-Hrs or the deletion constructs indicated were obtained by subcloning the respective cDNAs into the polylinker sites of pGEX-6P (Amersham Pharmacia). pGEX-2T-clathrin1–579 was a gift from Dr Jim Keen ( ).

Polymerase Chain Reaction:

Article Title: Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2
Article Snippet: To obtain the plasmid constructs used in the yeast two-hybrid (Y2H) assay, cDNAs encoding premature or mature LMP2 (obtained from the Y2H screen) and PSMB6 (synthetic gene obtained from Biovalley) were constructed in pGAD-GH (Clontech), a derivative of pGAD-GE. cDNAs coding for VSV M (serotype Indiana, strain Orsay) as well as all VSV M mutants were constructed in LexA (Clontech) ( ). .. Constructs of LMP2, PSMB6, M, MI96A, and dystonin in pCDNA3.1-myc or pCDNA3.1-HA (provided by D. Pasdeloup [ , ]) were generated by PCR.

Article Title: The 14-kDa Dynein Light Chain-Family Protein Dlc1 Is Required for Regular Oscillatory Nuclear Movement and Efficient Recombination during Meiotic Prophase in Fission Yeast
Article Snippet: A bait plasmid containing a segment of Kms1 protein (amino acid residues 202–607) fused to the Gal4 DNA-binding domain (Gal4BD) in pAS2.1 was used to screen an S. pombe cDNA library constructed with pGAD GH ( CLONTECH ). .. Various parts of Kms1 protein as well as Dlc1 were fused to the activation domain on pACT2 by using PCR-generated fragments.

Article Title: Rdp1, a Novel Zinc Finger Protein, Regulates the DNA Damage Response of rhp51+ from Schizosaccharomyces pombe
Article Snippet: The S. pombe cDNA expression library based on a 2μm- LEU2 plasmid, pGAD GH, was purchased from Clontech (catalog no. XL4000AA). .. The truncated cDNA sequence of rdp1 + was generated by PCR using oligonucleotides OR1F-Bam (5′-CACGGGATCCAACACTCCCACCGTAG-3′) and OR540R-EcoR (5′-GGGGTTGGAATCAGGCACTTGAC-3′) as primers and pGAD236 as the template.

Article Title: Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair
Article Snippet: The complete ORFs of RAD18, RAD5, UBC13 and MMS2 were cloned into the two-hybrid vectors pGAD424 and pGBT9 (Clontech) by PCR from genomic DNA. .. RAD6 was cloned into pAS2-1 and a derivative of pGAD GH (Clontech).

Article Title: Hrs recruits clathrin to early endosomes
Article Snippet: The Hrs constructs indicated were generated by PCR with mouse Hrs ( ) as the template. .. For use in the two-hybrid system, constructs were cloned into pLexA/pBTM116 ( ) as bait and pGAD GH (Clontech) as prey.

cDNA Library Assay:

Article Title: The 14-kDa Dynein Light Chain-Family Protein Dlc1 Is Required for Regular Oscillatory Nuclear Movement and Efficient Recombination during Meiotic Prophase in Fission Yeast
Article Snippet: .. A bait plasmid containing a segment of Kms1 protein (amino acid residues 202–607) fused to the Gal4 DNA-binding domain (Gal4BD) in pAS2.1 was used to screen an S. pombe cDNA library constructed with pGAD GH ( CLONTECH ). .. Various parts of Kms1 protein as well as Dlc1 were fused to the activation domain on pACT2 by using PCR-generated fragments.

Article Title: Live-Cell Imaging Reveals Multiple Interactions between Epstein-Barr Virus Nuclear Antigen 1 and Cellular Chromatin during Interphase and Mitosis
Article Snippet: .. A premade HeLa cell random cDNA library fused with GAL4 AD in pGAD-GH (Clontech) was used. ..

Article Title: Centrosomal Proteins CG-NAP and Kendrin Provide Microtubule Nucleation Sites by Anchoring ?-Tubulin Ring Complex
Article Snippet: .. The resultant plasmid was transformed to the yeast reporter strain AH109 together with a HeLa cDNA library fused to the Gal4 activation domain (Gal4ad) in pGAD GH ( Clontech ). .. Bacterial expression plasmids for GST-tagged CG-NAP3510–3829 and His6 -tagged calmodulin 2 were constructed by inserting the corresponding cDNA fragments into pGEX4T and pRSET (Invitrogen), respectively.

Article Title: Zyxin, a Regulator of Actin Filament Assembly, Targets the Mitotic Apparatus by Interacting with H-Warts/Lats1 Tumor Suppressor
Article Snippet: .. An L40 strain carrying pBTM116HA/h-warts (amino acids 394–675) was transformed with the HeLa cDNA library constructed in pGAD-GH (Clontech) by electroporation. ..

Plasmid Preparation:

Article Title: Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2
Article Snippet: .. To obtain the plasmid constructs used in the yeast two-hybrid (Y2H) assay, cDNAs encoding premature or mature LMP2 (obtained from the Y2H screen) and PSMB6 (synthetic gene obtained from Biovalley) were constructed in pGAD-GH (Clontech), a derivative of pGAD-GE. cDNAs coding for VSV M (serotype Indiana, strain Orsay) as well as all VSV M mutants were constructed in LexA (Clontech) ( ). ..

Article Title: The Growth Factor Granulin Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription
Article Snippet: .. The plasmids pGBT9, pGAD GH, and pACT2 and the S. cerevisiae strain CG1945 for yeast two-hybrid system experiments were from Clontech Laboratories Inc. Truncated versions of cyclin T1 were cloned into the S. cerevisiae vector pGBT9, and cyclin T1 amino acids 1 to 708 [cyclin T1(1-708)] was also cloned into pGAD GH to confirm the interactions. .. Full-length CDK9 was cloned into pACT2.

Article Title: The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1
Article Snippet: .. The cDNA of wild-type (WT) EED ( ) was inserted into the Eco RI and Not I sites of the Gal4 transcription activation domain vector pGAD3S2X, a modified version of the pGAD GH (Clontech) containing a Not I site in its polylinker. .. This resulted in a Gal4 activation domain AD-EED hybrid.

Article Title: The 14-kDa Dynein Light Chain-Family Protein Dlc1 Is Required for Regular Oscillatory Nuclear Movement and Efficient Recombination during Meiotic Prophase in Fission Yeast
Article Snippet: .. A bait plasmid containing a segment of Kms1 protein (amino acid residues 202–607) fused to the Gal4 DNA-binding domain (Gal4BD) in pAS2.1 was used to screen an S. pombe cDNA library constructed with pGAD GH ( CLONTECH ). .. Various parts of Kms1 protein as well as Dlc1 were fused to the activation domain on pACT2 by using PCR-generated fragments.

Article Title: Rdp1, a Novel Zinc Finger Protein, Regulates the DNA Damage Response of rhp51+ from Schizosaccharomyces pombe
Article Snippet: .. The S. pombe cDNA expression library based on a 2μm- LEU2 plasmid, pGAD GH, was purchased from Clontech (catalog no. XL4000AA). .. The truncated cDNA sequence of rdp1 + was generated by PCR using oligonucleotides OR1F-Bam (5′-CACGGGATCCAACACTCCCACCGTAG-3′) and OR540R-EcoR (5′-GGGGTTGGAATCAGGCACTTGAC-3′) as primers and pGAD236 as the template.

Article Title: Z-DNA-binding proteins can act as potent effectors of gene expression invivo
Article Snippet: .. Reporter vectors were constructed by using the pLacZi vector as a template and expression vectors were constructed by using either pACT2 or pGAD-GH (CLONTECH). ..

Article Title: Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair
Article Snippet: Plasmid mms2 Δ ::HIS3 was constructed by amplifying 511 bp of the upstream and 420 bp of the downstream flanking regions of MMS2 by PCR from genomic DNA and combining the fragments with a HIS3 cassette in pBluescript-SK(+) (Stratagene). rad18 Δ ::TRP1 contains 464 bp of the RAD18 upstream flanking region and the C-terminal part of the open reading frame (ORF; 520 bp) interrupted by the TRP1 locus in pBluescript-SK(+). rad30 Δ ::HIS 3 was constructed by inserting a 3076 bp PCR fragment containing the RAD30 gene with flanking regions into pBluescript-SK(+) and replacing the Stu I– Ehe I fragment, containing the entire ORF, with the HIS3 marker. rad5 Δ ::HIS3 was constructed similarly by amplification of the RAD5 ORF, insertion into pBluescript-SK(+) and replacement of the internal Bcl I– Bgl II fragment with HIS3 . .. RAD6 was cloned into pAS2-1 and a derivative of pGAD GH (Clontech).

Article Title: A Chimeric Protein Containing the N Terminus of the Adeno-Associated Virus Rep Protein Recognizes Its Target Site in an In Vivo Assay
Article Snippet: .. An Aat II- Hin dIII fragment of pGAD GH (Clontech) containing the full-length promoter was subcloned into the pG expression plasmid series to replace a corresponding fragment containing a truncated version of the ADH1 promoter, giving rise to pADH.Rep68, pADH.Rep78, pADH.Rep.AD, pADH.Rep.LZ.AD, pADH.Rep.TZ.AD, and pADH.Rep.TZ. .. Plasmid pADH.Keratin was kindly provided by Jeanette Ducut.

Article Title: Centrosomal Proteins CG-NAP and Kendrin Provide Microtubule Nucleation Sites by Anchoring ?-Tubulin Ring Complex
Article Snippet: .. The resultant plasmid was transformed to the yeast reporter strain AH109 together with a HeLa cDNA library fused to the Gal4 activation domain (Gal4ad) in pGAD GH ( Clontech ). .. Bacterial expression plasmids for GST-tagged CG-NAP3510–3829 and His6 -tagged calmodulin 2 were constructed by inserting the corresponding cDNA fragments into pGEX4T and pRSET (Invitrogen), respectively.

Article Title: Zyxin, a Regulator of Actin Filament Assembly, Targets the Mitotic Apparatus by Interacting with H-Warts/Lats1 Tumor Suppressor
Article Snippet: An L40 strain carrying pBTM116HA/h-warts (amino acids 394–675) was transformed with the HeLa cDNA library constructed in pGAD-GH (Clontech) by electroporation. .. Plasmids harboring cDNA were recovered from positive colonies and the nucleotide sequence of plasmid DNA which conferred the LacZ+ phenotype on L40 containing pBTM116HA/h-warts (396–657) were determined.

Article Title: Hrs recruits clathrin to early endosomes
Article Snippet: Paragraph title: Plasmid constructs ... For use in the two-hybrid system, constructs were cloned into pLexA/pBTM116 ( ) as bait and pGAD GH (Clontech) as prey.

Recombinant:

Article Title: The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1
Article Snippet: The sequence of the construct was verified by DNA sequencing, and the expression of recombinant fusion protein in yeast was confirmed by gel electrophoresis and immunoblot analysis with anti-IN polyclonal antibody as described below. (ii) Generation of the pGAD-EED (Fig. ). .. The cDNA of wild-type (WT) EED ( ) was inserted into the Eco RI and Not I sites of the Gal4 transcription activation domain vector pGAD3S2X, a modified version of the pGAD GH (Clontech) containing a Not I site in its polylinker.

Two Hybrid Screening:

Article Title: MCAF Mediates MBD1-Dependent Transcriptional Repression
Article Snippet: Paragraph title: Yeast two-hybrid screening. ... Yeast strain CG-1945 carrying pAS2-1-TRD of MBD1 (amino acids 529 to 592 [isoform v1] or 473 to 536 [isoform v3]) ( ) was transformed with the HeLa cDNA libraries constructed in pGAD-GH (Clontech).

Article Title: Live-Cell Imaging Reveals Multiple Interactions between Epstein-Barr Virus Nuclear Antigen 1 and Cellular Chromatin during Interphase and Mitosis
Article Snippet: Paragraph title: Yeast two-hybrid screening. ... A premade HeLa cell random cDNA library fused with GAL4 AD in pGAD-GH (Clontech) was used.

Article Title: Centrosomal Proteins CG-NAP and Kendrin Provide Microtubule Nucleation Sites by Anchoring ?-Tubulin Ring Complex
Article Snippet: Paragraph title: Yeast Two-Hybrid Screening and Interaction Analysis ... The resultant plasmid was transformed to the yeast reporter strain AH109 together with a HeLa cDNA library fused to the Gal4 activation domain (Gal4ad) in pGAD GH ( Clontech ).

Article Title: Zyxin, a Regulator of Actin Filament Assembly, Targets the Mitotic Apparatus by Interacting with H-Warts/Lats1 Tumor Suppressor
Article Snippet: Paragraph title: Yeast Two-Hybrid Screening ... An L40 strain carrying pBTM116HA/h-warts (amino acids 394–675) was transformed with the HeLa cDNA library constructed in pGAD-GH (Clontech) by electroporation.

Activation Assay:

Article Title: The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1
Article Snippet: .. The cDNA of wild-type (WT) EED ( ) was inserted into the Eco RI and Not I sites of the Gal4 transcription activation domain vector pGAD3S2X, a modified version of the pGAD GH (Clontech) containing a Not I site in its polylinker. .. This resulted in a Gal4 activation domain AD-EED hybrid.

Article Title: The 14-kDa Dynein Light Chain-Family Protein Dlc1 Is Required for Regular Oscillatory Nuclear Movement and Efficient Recombination during Meiotic Prophase in Fission Yeast
Article Snippet: A bait plasmid containing a segment of Kms1 protein (amino acid residues 202–607) fused to the Gal4 DNA-binding domain (Gal4BD) in pAS2.1 was used to screen an S. pombe cDNA library constructed with pGAD GH ( CLONTECH ). .. Various parts of Kms1 protein as well as Dlc1 were fused to the activation domain on pACT2 by using PCR-generated fragments.

Article Title: Centrosomal Proteins CG-NAP and Kendrin Provide Microtubule Nucleation Sites by Anchoring ?-Tubulin Ring Complex
Article Snippet: .. The resultant plasmid was transformed to the yeast reporter strain AH109 together with a HeLa cDNA library fused to the Gal4 activation domain (Gal4ad) in pGAD GH ( Clontech ). .. Bacterial expression plasmids for GST-tagged CG-NAP3510–3829 and His6 -tagged calmodulin 2 were constructed by inserting the corresponding cDNA fragments into pGEX4T and pRSET (Invitrogen), respectively.

Marker:

Article Title: Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair
Article Snippet: Plasmid mms2 Δ ::HIS3 was constructed by amplifying 511 bp of the upstream and 420 bp of the downstream flanking regions of MMS2 by PCR from genomic DNA and combining the fragments with a HIS3 cassette in pBluescript-SK(+) (Stratagene). rad18 Δ ::TRP1 contains 464 bp of the RAD18 upstream flanking region and the C-terminal part of the open reading frame (ORF; 520 bp) interrupted by the TRP1 locus in pBluescript-SK(+). rad30 Δ ::HIS 3 was constructed by inserting a 3076 bp PCR fragment containing the RAD30 gene with flanking regions into pBluescript-SK(+) and replacing the Stu I– Ehe I fragment, containing the entire ORF, with the HIS3 marker. rad5 Δ ::HIS3 was constructed similarly by amplification of the RAD5 ORF, insertion into pBluescript-SK(+) and replacement of the internal Bcl I– Bgl II fragment with HIS3 . .. RAD6 was cloned into pAS2-1 and a derivative of pGAD GH (Clontech).

Staining:

Article Title: The trans-Golgi network GRIP-domain proteins form ?-helical homodimers
Article Snippet: pHybLex-GCC88, pHybLex-GCC88aa1-330 , pHybLex-GCC88aa331-775 , pGAD-GCC88 and pGAD-GCC88aa441-775 were obtained by subcloning the full-length [ ] and partial GCC88 cDNAs, isolated from full-length GCC88 cDNA by digestion with restriction enzymes, into the polylinker sites of pHybLex/Zeo (LexA-binding domain; Invitrogen) and pGAD GH (GAL4 activation domain; Clontech, Palo Alto, CA, U.S.A.) respectively. .. The pHybLex/Zeo and pGAD GH constructs were co-transformed in to the Saccharomyces cerevisiae reporter stain L40 [ ] using a lithium acetate method [ ].

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91
    TaKaRa pgad gh
    The matrix protein of VSV does not interact with PSMB6. (A) cDNAs encoding pre-LMP2 as well as pre-PSMB6 and mature PSMB6 were constructed in <t>pGAD</t> and used in <t>Y2H</t> assays against a pLEX VSV M construct. The empty pGAD (AD) or pLEX (LEX BD) vector was used as a negative control. The interaction was evaluated by quantification of β-galactosidase activity in liquid yeast cultures (see the Materials and Methods section). Bars represent standard deviations of the means from three independent experiments performed in triplicate. (B) Coimmunoprecipitation of M and PSMB6. N2A cells were transfected or not transfected (Non TF) for 24 h with plasmids carrying myc-pre-LMP2 (positive control), myc-pre-PSMB6, myc-mature-PSMB6, or myc-dystonin as a negative control and then infected with VSV (4 h; MOI, 3) before preparation of cell lysates. Following immunoprecipitation with an anti-M antibody or anti-myc antibody 9E10, cell extracts (inputs) and immune complexes (immunoprecipitates [IP]) were separated by SDS-PAGE and analyzed by Western blotting using anti-myc antibody 9E10 or M-specific antibody to reveal the presence of the different myc-LMP2, myc-PSMB6, and myc-dystonin constructs and M.
    Pgad Gh, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgad gh/product/TaKaRa
    Average 91 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    pgad gh - by Bioz Stars, 2020-02
    91/100 stars
      Buy from Supplier

    83
    TaKaRa activation domain vector pgad gh
    Kir2 channels but not Kir3 channels interact with <t>PDZ</t> domains 1–3 in the Y2H system. A , C termini of the Kir channels listed were used as bait in pGBT9 to test the interaction with PDZ1–3 of PSD-95 in <t>pGAD-GH.</t> The NR2B subunit and the Kv1.4 C termini were used as positive controls. The consensus sequence X-S/T-X-I/V is shown in bold letters . Numbers on the left denote amino acid positions of C-terminal fragments. B , Cross-mutational analysis of Kir2.1 and Kir3.2 mutants. See Results for details. Colony growth was controlled after 4 d.
    Activation Domain Vector Pgad Gh, supplied by TaKaRa, used in various techniques. Bioz Stars score: 83/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/activation domain vector pgad gh/product/TaKaRa
    Average 83 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    activation domain vector pgad gh - by Bioz Stars, 2020-02
    83/100 stars
      Buy from Supplier

    94
    TaKaRa pgad gh vector
    The RNA-supplemented two-hybrid system. ( A ) The DNA-binding-domain hybrid is composed of the <t>GAL4</t> DNA binding domain (DBD) fused to the ΔC27 mutant of human SLBP. The RNA component contains the RNase P leader at the 5′ end and two MS2-binding sites in the center, followed by the histone stem–loop. In the diagram only one MS2-binding site is depicted, and the RNase P leader is omitted. Protein X fused to the GAL4 activation domain (AD) interacts with the SLBP/RNA complex. The interaction brings the GAL4 AD into proximity to the promoter, resulting in activation of the HIS3 and LacZ reporter genes controlled by the GAL4-binding site. ( B ) Clones isolated in the RNA-supplemented two-hybrid system interact with the SLBP/SL complex but not with SLBP. Expression of two reporter genes, HIS3 and LacZ , was determined by testing the ability of yeast cells to grow on selective medium and by assaying β-galactosidase activity. To determine expression of the HIS3 gene, a suspension of yeast containing the library plasmid <t>pGAD</t> GH with the indicated cDNA insert, and the bait plasmid expressing SLBP with ( left , lanes 1 – 3 ) or without stem–loop RNA ( right , lanes 4 – 6 ), was spotted on medium with histidine (lanes 1,4 ) and on medium lacking histidine containing 10 mM 3-AT (lanes 2,5 ). hZFP100 and two other clones (8 and 22) isolated by the RNA-supplemented two-hybrid system are shown. Clone 8 is a putative 36-kD protein and clone 22 is ribosomal protein S15a. The ability of each clone to interact with the SLBP/RNA complex (lane 3 ) and free SLBP (lane 6 ) was confirmed by analysis of expression of the LacZ gene using a qualitative blue/white assay. After a 2-h incubation, cell suspensions were spotted on a white background and photographed. ( C ) The hSLBP was replaced in the bait construct shown in A with the Drosophila SLBP, and the ability of the three clones isolated in the initial screen to grow in the presence of 10 mM 3-AT was determined.
    Pgad Gh Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgad gh vector/product/TaKaRa
    Average 94 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    pgad gh vector - by Bioz Stars, 2020-02
    94/100 stars
      Buy from Supplier

    Image Search Results


    The matrix protein of VSV does not interact with PSMB6. (A) cDNAs encoding pre-LMP2 as well as pre-PSMB6 and mature PSMB6 were constructed in pGAD and used in Y2H assays against a pLEX VSV M construct. The empty pGAD (AD) or pLEX (LEX BD) vector was used as a negative control. The interaction was evaluated by quantification of β-galactosidase activity in liquid yeast cultures (see the Materials and Methods section). Bars represent standard deviations of the means from three independent experiments performed in triplicate. (B) Coimmunoprecipitation of M and PSMB6. N2A cells were transfected or not transfected (Non TF) for 24 h with plasmids carrying myc-pre-LMP2 (positive control), myc-pre-PSMB6, myc-mature-PSMB6, or myc-dystonin as a negative control and then infected with VSV (4 h; MOI, 3) before preparation of cell lysates. Following immunoprecipitation with an anti-M antibody or anti-myc antibody 9E10, cell extracts (inputs) and immune complexes (immunoprecipitates [IP]) were separated by SDS-PAGE and analyzed by Western blotting using anti-myc antibody 9E10 or M-specific antibody to reveal the presence of the different myc-LMP2, myc-PSMB6, and myc-dystonin constructs and M.

    Journal: Journal of Virology

    Article Title: Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2

    doi: 10.1128/JVI.01753-15

    Figure Lengend Snippet: The matrix protein of VSV does not interact with PSMB6. (A) cDNAs encoding pre-LMP2 as well as pre-PSMB6 and mature PSMB6 were constructed in pGAD and used in Y2H assays against a pLEX VSV M construct. The empty pGAD (AD) or pLEX (LEX BD) vector was used as a negative control. The interaction was evaluated by quantification of β-galactosidase activity in liquid yeast cultures (see the Materials and Methods section). Bars represent standard deviations of the means from three independent experiments performed in triplicate. (B) Coimmunoprecipitation of M and PSMB6. N2A cells were transfected or not transfected (Non TF) for 24 h with plasmids carrying myc-pre-LMP2 (positive control), myc-pre-PSMB6, myc-mature-PSMB6, or myc-dystonin as a negative control and then infected with VSV (4 h; MOI, 3) before preparation of cell lysates. Following immunoprecipitation with an anti-M antibody or anti-myc antibody 9E10, cell extracts (inputs) and immune complexes (immunoprecipitates [IP]) were separated by SDS-PAGE and analyzed by Western blotting using anti-myc antibody 9E10 or M-specific antibody to reveal the presence of the different myc-LMP2, myc-PSMB6, and myc-dystonin constructs and M.

    Article Snippet: To obtain the plasmid constructs used in the yeast two-hybrid (Y2H) assay, cDNAs encoding premature or mature LMP2 (obtained from the Y2H screen) and PSMB6 (synthetic gene obtained from Biovalley) were constructed in pGAD-GH (Clontech), a derivative of pGAD-GE. cDNAs coding for VSV M (serotype Indiana, strain Orsay) as well as all VSV M mutants were constructed in LexA (Clontech) ( ).

    Techniques: Construct, Plasmid Preparation, Negative Control, Activity Assay, Transfection, Positive Control, Infection, Immunoprecipitation, SDS Page, Western Blot

    The matrix protein of VSV interacts with LMP2. (A) cDNAs encoding pre-LMP2, mature LMP2, and the 20-amino-acid N-terminal propeptide were constructed in pGAD and used in Y2H assays against a pLEX VSV M construct. The empty pGAD (AD) or pLEX (LEX BD) vector was used as a negative control. The interaction was evaluated by quantification of β-galactosidase activity in liquid yeast cultures as described in the Materials and Methods section. Bars represent standard deviations of the mean from three independent experiments performed in triplicate. *, P

    Journal: Journal of Virology

    Article Title: Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2

    doi: 10.1128/JVI.01753-15

    Figure Lengend Snippet: The matrix protein of VSV interacts with LMP2. (A) cDNAs encoding pre-LMP2, mature LMP2, and the 20-amino-acid N-terminal propeptide were constructed in pGAD and used in Y2H assays against a pLEX VSV M construct. The empty pGAD (AD) or pLEX (LEX BD) vector was used as a negative control. The interaction was evaluated by quantification of β-galactosidase activity in liquid yeast cultures as described in the Materials and Methods section. Bars represent standard deviations of the mean from three independent experiments performed in triplicate. *, P

    Article Snippet: To obtain the plasmid constructs used in the yeast two-hybrid (Y2H) assay, cDNAs encoding premature or mature LMP2 (obtained from the Y2H screen) and PSMB6 (synthetic gene obtained from Biovalley) were constructed in pGAD-GH (Clontech), a derivative of pGAD-GE. cDNAs coding for VSV M (serotype Indiana, strain Orsay) as well as all VSV M mutants were constructed in LexA (Clontech) ( ).

    Techniques: Construct, Plasmid Preparation, Negative Control, Activity Assay

    Fig. 3. The C-terminus of Hrs binds clathrin TD. ( A ). This illustrates the existence of a potential clathrin-binding motif within residues 770–775 of Hrs. Hrs is the only protein that has the clathrin box motif at the very C-terminus. (B–E) Interaction of Hrs with clathrin. ( B and C ) L40 reporter yeast cells were transformed with bait constructs in pLexA and prey constructs in pGAD. Reporter β-galactosidase activities (in arbitrary units) indicate binding and are represented as mean values of two independent experiments performed in duplicate. Error bars denote ± SEM. In (B), a clathrin triskelion (consisting of three heavy chains) is illustrated, with the terminal domain (TD), distal domain (DD) and hub domain (HD) indicated. ( D ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3) were immobilized on glutathione–Sepharose beads and incubated with pig brain cytosol. The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin with anti-clathrin heavy chain antibodies (upper panel). ( E ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3), were immobilized on glutathione–Sepharose beads and incubated with purified recombinant clathrin terminal domain (TD 1–579 ). The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin-TD 1–579 with anti-clathrin heavy chain (upper panel). Lane 4 represents the total amount of recombinant clathrin-TD 1–579 added to the beads.

    Journal: The EMBO Journal

    Article Title: Hrs recruits clathrin to early endosomes

    doi: 10.1093/emboj/20.17.5008

    Figure Lengend Snippet: Fig. 3. The C-terminus of Hrs binds clathrin TD. ( A ). This illustrates the existence of a potential clathrin-binding motif within residues 770–775 of Hrs. Hrs is the only protein that has the clathrin box motif at the very C-terminus. (B–E) Interaction of Hrs with clathrin. ( B and C ) L40 reporter yeast cells were transformed with bait constructs in pLexA and prey constructs in pGAD. Reporter β-galactosidase activities (in arbitrary units) indicate binding and are represented as mean values of two independent experiments performed in duplicate. Error bars denote ± SEM. In (B), a clathrin triskelion (consisting of three heavy chains) is illustrated, with the terminal domain (TD), distal domain (DD) and hub domain (HD) indicated. ( D ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3) were immobilized on glutathione–Sepharose beads and incubated with pig brain cytosol. The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin with anti-clathrin heavy chain antibodies (upper panel). ( E ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3), were immobilized on glutathione–Sepharose beads and incubated with purified recombinant clathrin terminal domain (TD 1–579 ). The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin-TD 1–579 with anti-clathrin heavy chain (upper panel). Lane 4 represents the total amount of recombinant clathrin-TD 1–579 added to the beads.

    Article Snippet: For use in the two-hybrid system, constructs were cloned into pLexA/pBTM116 ( ) as bait and pGAD GH (Clontech) as prey.

    Techniques: Binding Assay, Transformation Assay, Construct, Recombinant, Incubation, Centrifugation, SDS Page, Staining, Purification

    Kir2 channels but not Kir3 channels interact with PDZ domains 1–3 in the Y2H system. A , C termini of the Kir channels listed were used as bait in pGBT9 to test the interaction with PDZ1–3 of PSD-95 in pGAD-GH. The NR2B subunit and the Kv1.4 C termini were used as positive controls. The consensus sequence X-S/T-X-I/V is shown in bold letters . Numbers on the left denote amino acid positions of C-terminal fragments. B , Cross-mutational analysis of Kir2.1 and Kir3.2 mutants. See Results for details. Colony growth was controlled after 4 d.

    Journal: The Journal of Neuroscience

    Article Title: Neuronal Inwardly Rectifying K+ Channels Differentially Couple to PDZ Proteins of the PSD-95/SAP90 Family

    doi: 10.1523/JNEUROSCI.20-01-00156.2000

    Figure Lengend Snippet: Kir2 channels but not Kir3 channels interact with PDZ domains 1–3 in the Y2H system. A , C termini of the Kir channels listed were used as bait in pGBT9 to test the interaction with PDZ1–3 of PSD-95 in pGAD-GH. The NR2B subunit and the Kv1.4 C termini were used as positive controls. The consensus sequence X-S/T-X-I/V is shown in bold letters . Numbers on the left denote amino acid positions of C-terminal fragments. B , Cross-mutational analysis of Kir2.1 and Kir3.2 mutants. See Results for details. Colony growth was controlled after 4 d.

    Article Snippet: PDZ domains 1–3 of PSD-95/SAP90 (aa 1–401) were inserted into the activation domain vector pGAD-GH (Clontech).

    Techniques: Sequencing

    The RNA-supplemented two-hybrid system. ( A ) The DNA-binding-domain hybrid is composed of the GAL4 DNA binding domain (DBD) fused to the ΔC27 mutant of human SLBP. The RNA component contains the RNase P leader at the 5′ end and two MS2-binding sites in the center, followed by the histone stem–loop. In the diagram only one MS2-binding site is depicted, and the RNase P leader is omitted. Protein X fused to the GAL4 activation domain (AD) interacts with the SLBP/RNA complex. The interaction brings the GAL4 AD into proximity to the promoter, resulting in activation of the HIS3 and LacZ reporter genes controlled by the GAL4-binding site. ( B ) Clones isolated in the RNA-supplemented two-hybrid system interact with the SLBP/SL complex but not with SLBP. Expression of two reporter genes, HIS3 and LacZ , was determined by testing the ability of yeast cells to grow on selective medium and by assaying β-galactosidase activity. To determine expression of the HIS3 gene, a suspension of yeast containing the library plasmid pGAD GH with the indicated cDNA insert, and the bait plasmid expressing SLBP with ( left , lanes 1 – 3 ) or without stem–loop RNA ( right , lanes 4 – 6 ), was spotted on medium with histidine (lanes 1,4 ) and on medium lacking histidine containing 10 mM 3-AT (lanes 2,5 ). hZFP100 and two other clones (8 and 22) isolated by the RNA-supplemented two-hybrid system are shown. Clone 8 is a putative 36-kD protein and clone 22 is ribosomal protein S15a. The ability of each clone to interact with the SLBP/RNA complex (lane 3 ) and free SLBP (lane 6 ) was confirmed by analysis of expression of the LacZ gene using a qualitative blue/white assay. After a 2-h incubation, cell suspensions were spotted on a white background and photographed. ( C ) The hSLBP was replaced in the bait construct shown in A with the Drosophila SLBP, and the ability of the three clones isolated in the initial screen to grow in the presence of 10 mM 3-AT was determined.

    Journal: Genes & Development

    Article Title: A novel zinc finger protein is associated with U7 snRNP and interacts with the stem-loop binding protein in the histone pre-mRNP to stimulate 3?-end processing

    doi: 10.1101/gad.932302

    Figure Lengend Snippet: The RNA-supplemented two-hybrid system. ( A ) The DNA-binding-domain hybrid is composed of the GAL4 DNA binding domain (DBD) fused to the ΔC27 mutant of human SLBP. The RNA component contains the RNase P leader at the 5′ end and two MS2-binding sites in the center, followed by the histone stem–loop. In the diagram only one MS2-binding site is depicted, and the RNase P leader is omitted. Protein X fused to the GAL4 activation domain (AD) interacts with the SLBP/RNA complex. The interaction brings the GAL4 AD into proximity to the promoter, resulting in activation of the HIS3 and LacZ reporter genes controlled by the GAL4-binding site. ( B ) Clones isolated in the RNA-supplemented two-hybrid system interact with the SLBP/SL complex but not with SLBP. Expression of two reporter genes, HIS3 and LacZ , was determined by testing the ability of yeast cells to grow on selective medium and by assaying β-galactosidase activity. To determine expression of the HIS3 gene, a suspension of yeast containing the library plasmid pGAD GH with the indicated cDNA insert, and the bait plasmid expressing SLBP with ( left , lanes 1 – 3 ) or without stem–loop RNA ( right , lanes 4 – 6 ), was spotted on medium with histidine (lanes 1,4 ) and on medium lacking histidine containing 10 mM 3-AT (lanes 2,5 ). hZFP100 and two other clones (8 and 22) isolated by the RNA-supplemented two-hybrid system are shown. Clone 8 is a putative 36-kD protein and clone 22 is ribosomal protein S15a. The ability of each clone to interact with the SLBP/RNA complex (lane 3 ) and free SLBP (lane 6 ) was confirmed by analysis of expression of the LacZ gene using a qualitative blue/white assay. After a 2-h incubation, cell suspensions were spotted on a white background and photographed. ( C ) The hSLBP was replaced in the bait construct shown in A with the Drosophila SLBP, and the ability of the three clones isolated in the initial screen to grow in the presence of 10 mM 3-AT was determined.

    Article Snippet: This strain harboring the pGBT/SLBP/SLWT plasmid was used to screen a GAL4 AD/HeLa cDNA fusion library constructed in the pGAD GH vector (Clontech).

    Techniques: Binding Assay, Mutagenesis, Activation Assay, Clone Assay, Isolation, Expressing, Activity Assay, Plasmid Preparation, Incubation, Construct