Journal: Retrovirology

Article Title: NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

doi: 10.1186/s12977-014-0067-y

Figure Lengend Snippet: Interaction between HIV-1 Tat and NUCKS1. (A) Tat-expressing plasmid cloned into pGBKT7 and NUCKS1 selected from human thymus cDNA library cloned into pACT2 (Clontech) was transformed into the yeast strain PBN204. The transformants were grown on an SD plate lacking adenosine and uracil for 48 h. Murine p53 and SV40 large T antigens were used as a positive control. Two panels show yeast colonies showing the interaction of Tat and NUCKS1. (B) Ectopically expressed Tat interacts with ectopically expressed NUCKS1 in HEK293 cells. Two micrograms of the Flag–Tat, Flag-Vpr and Flag-Nef expression vector were cotransfected with the V5–NUCKS1 expression vector into HEK293 cells cultured in 100 mm plates. Forty-eight hours after transfection, cell lysates were immunoprecipitated with an anti-Flag monoclonal antibody (M2). Immunoprecipitates were analyzed by Western blotting using an HRP-conjugated anti-V5 monoclonal antibody. The Flag- or V5-tagged pcDNA3 plasmid was added to equalize the total amounts of DNA. (C) Coimmunoprecipitation assay between endogenous NUCKS1 and ectopically expressed Tat protein. The lysate from Tat-expressing HEK293T cells was immunoprecipitated with anti-Flag and -Pol II antibodies, and the interaction was assessed with Western blotting using anti-NUCKS1 or anti-Flag antibody.

Article Snippet: Figure 1 Interaction between HIV-1 Tat and NUCKS1. (A) Tat-expressing plasmid cloned into pGBKT7 and NUCKS1 selected from human thymus cDNA library cloned into pACT2 (Clontech) was transformed into the yeast strain PBN204.

Techniques: Expressing, Plasmid Preparation, Clone Assay, cDNA Library Assay, Transformation Assay, Positive Control, Cell Culture, Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay

Journal: Retrovirology

Article Title: NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

doi: 10.1186/s12977-014-0067-y

Figure Lengend Snippet: Interaction between HIV-1 Tat and NUCKS1. (A) Tat-expressing plasmid cloned into pGBKT7 and NUCKS1 selected from human thymus cDNA library cloned into pACT2 (Clontech) was transformed into the yeast strain PBN204. The transformants were grown on an SD plate lacking adenosine and uracil for 48 h. Murine p53 and SV40 large T antigens were used as a positive control. Two panels show yeast colonies showing the interaction of Tat and NUCKS1. (B) Ectopically expressed Tat interacts with ectopically expressed NUCKS1 in HEK293 cells. Two micrograms of the Flag–Tat, Flag-Vpr and Flag-Nef expression vector were cotransfected with the V5–NUCKS1 expression vector into HEK293 cells cultured in 100 mm plates. Forty-eight hours after transfection, cell lysates were immunoprecipitated with an anti-Flag monoclonal antibody (M2). Immunoprecipitates were analyzed by Western blotting using an HRP-conjugated anti-V5 monoclonal antibody. The Flag- or V5-tagged pcDNA3 plasmid was added to equalize the total amounts of DNA. (C) Coimmunoprecipitation assay between endogenous NUCKS1 and ectopically expressed Tat protein. The lysate from Tat-expressing HEK293T cells was immunoprecipitated with anti-Flag and -Pol II antibodies, and the interaction was assessed with Western blotting using anti-NUCKS1 or anti-Flag antibody.

Article Snippet: Figure 1 Interaction between HIV-1 Tat and NUCKS1. (A) Tat-expressing plasmid cloned into pGBKT7 and NUCKS1 selected from human thymus cDNA library cloned into pACT2 (Clontech) was transformed into the yeast strain PBN204.

Techniques: Expressing, Plasmid Preparation, Clone Assay, cDNA Library Assay, Transformation Assay, Positive Control, Cell Culture, Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay

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: The Journal of Biological Chemistry

Article Title: GREBP, a cGMP-response Element-binding Protein Repressing the Transcription of Natriuretic Peptide Receptor 1 (NPR1/GCA) *

doi: 10.1074/jbc.M109.061622

Figure Lengend Snippet: Identification of a cGMP-response element-binding protein. A, yeast one-hybrid screening of a human kidney cDNA library; the putative binding protein interacts with cGMP-RE (present in three copies) to activate the reporter gene via GAL4AD. B, full-length mRNA determination was by primer extension. HEK293 total RNA was retrotranscribed using a radiolabeled primer targeting GREBP mRNA and run beside pACT2-GREBP plasmid sequencing with the same radioactive primer. C, full-length GREBP mRNA was confirmed by RT-PCR with a specific primer targeting upstream and downstream regions of the transcription start site. The positive control was HEK293 DNA, whereas retrotranscribed HEK293 RNA was studied by −25/−5 and +1/+20 RT-PCR. D, nucleotide and amino acid sequences of GREBP. The mRNA destabilization motif is double underlined. Basic residues (framed) constitute 20% of GREBP (23 of 115) and are distributed along the whole sequence. Boldface residues are those with high possibility of serine phosphorylation (>0.800:1.00), and threonine could be strongly phosphorylated (>0.900:1.00) by serine/threonine kinase, including PKG.

Article Snippet: Yeast One-hybrid Screening of a Human Kidney cDNA Library A human kidney cDNA library was obtained from Clontech (Mountain View, CA) as pACT2 plasmids already transformed in the bacterial strain BNN132.

Techniques: Binding Assay, cDNA Library Assay, Plasmid Preparation, Sequencing, Reverse Transcription Polymerase Chain Reaction, Positive Control

Journal: Biology Open

Article Title: TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

doi: 10.1242/bio.20131065

Figure Lengend Snippet: ( A ) Yeast co-transformation of pACT-2-TCTEX1D4 with PPP1CA, PPP1CC1, PPP1CC2 or PPP1CC2end in pAS2-1 vector, using the Li-Ac method. For negative and positive controls pAS2-1/pACT-2 and pVA3/pTD1 vectors were used, respectively. ( B ) Western blot showing that TCTEX1D4 binds to and co-immunoprecipitates PPP1CC in COS-7 cells transfected with Myc-TCTEX1D4. Non-transfected and transfected COS-7 cells were used as negative and positive controls, respectively. ( C ) Bacterial extracts expressing pET-TCTEX1D4, pET-TCTEX1D4-AAAA, pET-TCTEX1D4-NT (NT) and pET-TCTEX1D4-CT (CT) were run in an SDS-PAGE gel and overlaid with purified PPP1CC isoforms. pET vector alone was used as negative control and pET-NEK2C, a known PIP, was used as positive control. All experiments were repeated at least three times.

Article Snippet: This construct was used in immunoprecipitation and immunofluorescence. pACT-TCTEX1D4 – TCTEX1D4 cDNA was digested from Myc-TCTEX1D4 with Eco RI /XhoI , and inserted into Eco RI/ XhoI digested pACT-2 (Clontech, Saint Germain-en-Laye, France), using standard molecular biology procedures.

Techniques: Transformation Assay, Plasmid Preparation, Western Blot, Transfection, Expressing, SDS Page, Purification, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: The Nuclear Envelope Protein, LAP1B, Is a Novel Protein Phosphatase 1 Substrate

doi: 10.1371/journal.pone.0076788

Figure Lengend Snippet: Full-length LAP1B and its deletion mutants cloned in the pACT2 vector are represented. The red boxes represent the RVxF motifs present in the constructs, the green boxes correspond to the SILK motif, and the yellow boxes represent the transmembrane domain. A- Positive interactions were observed between LAP1B and PP1α, PP1γ1 and PP1γ2, but not with the C-terminus of PP1γ2 (PP1γ2End). B- PP1γ1 interacted with LAP1B-BM1 and BM1/2 but not with LAP1B-BM2 and BM3. C- Association of murine p53 and SV40 large T antigen was used as positive control (+) and co-transformation of pAS2-1 and pACT2 vectors as negative control (-).

Article Snippet: The amplified fragments were subcloned into the EcoRI/XhoI restriction sites of the pACT2 vector (Clontech).

Techniques: Clone Assay, Plasmid Preparation, Construct, Positive Control, Transformation Assay, Negative Control