Structured Review

TaKaRa pcdna
Pcdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcdna/product/TaKaRa
Average 92 stars, based on 7 article reviews
Price from $9.99 to $1999.99
pcdna - by Bioz Stars, 2020-09
92/100 stars

Images

Related Articles

Polymerase Chain Reaction:

Article Title: Interleukin-2 (IL-2) of flounder (Paralichthys olivaceus) as immune adjuvant enhance the immune effects of E. tarda subunit vaccine OmpV against Edwardsiellosis.
Article Snippet: .. In our previous study, we cloned and explored the biological functions of flounder (Paralichthys olivaceus) interleukin-2 (poIL-2), and showed that poIL-2 might have adjuvant potential for fish vaccines. .. In our previous study, we cloned and explored the biological functions of flounder (Paralichthys olivaceus) interleukin-2 (poIL-2), and showed that poIL-2 might have adjuvant potential for fish vaccines.

Construct:

Article Title: Interleukin-2 (IL-2) of flounder (Paralichthys olivaceus) as immune adjuvant enhance the immune effects of E. tarda subunit vaccine OmpV against Edwardsiellosis.
Article Snippet: .. In our previous study, we cloned and explored the biological functions of flounder (Paralichthys olivaceus) interleukin-2 (poIL-2), and showed that poIL-2 might have adjuvant potential for fish vaccines. .. In our previous study, we cloned and explored the biological functions of flounder (Paralichthys olivaceus) interleukin-2 (poIL-2), and showed that poIL-2 might have adjuvant potential for fish vaccines.

TA Cloning:

Article Title: Interleukin-2 (IL-2) of flounder (Paralichthys olivaceus) as immune adjuvant enhance the immune effects of E. tarda subunit vaccine OmpV against Edwardsiellosis.
Article Snippet: .. In our previous study, we cloned and explored the biological functions of flounder (Paralichthys olivaceus) interleukin-2 (poIL-2), and showed that poIL-2 might have adjuvant potential for fish vaccines. .. In our previous study, we cloned and explored the biological functions of flounder (Paralichthys olivaceus) interleukin-2 (poIL-2), and showed that poIL-2 might have adjuvant potential for fish vaccines.

Plasmid Preparation:

Article Title: Interleukin-2 (IL-2) of flounder (Paralichthys olivaceus) as immune adjuvant enhance the immune effects of E. tarda subunit vaccine OmpV against Edwardsiellosis.
Article Snippet: .. In our previous study, we cloned and explored the biological functions of flounder (Paralichthys olivaceus) interleukin-2 (poIL-2), and showed that poIL-2 might have adjuvant potential for fish vaccines. .. In our previous study, we cloned and explored the biological functions of flounder (Paralichthys olivaceus) interleukin-2 (poIL-2), and showed that poIL-2 might have adjuvant potential for fish vaccines.

Purification:

Article Title: Interleukin-2 (IL-2) of flounder (Paralichthys olivaceus) as immune adjuvant enhance the immune effects of E. tarda subunit vaccine OmpV against Edwardsiellosis.
Article Snippet: .. In our previous study, we cloned and explored the biological functions of flounder (Paralichthys olivaceus) interleukin-2 (poIL-2), and showed that poIL-2 might have adjuvant potential for fish vaccines. .. In our previous study, we cloned and explored the biological functions of flounder (Paralichthys olivaceus) interleukin-2 (poIL-2), and showed that poIL-2 might have adjuvant potential for fish vaccines.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    TaKaRa pcdna il 4
    RT-PCR results for the transfected cells. M, DL2000 DNA marker; Lanes 1, 3, and 5, RT-PCR products from cells transfected with pcDNA3.1(+); Lane 2, RT-PCR product from cells transfected with <t>pcDNA-IL-2;</t> Lane 4, RT-PCR product from cells transfected with <t>pcDNA-IL-4;</t> Lane 6, RT-PCR product from cells transfected with pcDNA-ORF5.
    Pcdna Il 4, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna il 4/product/TaKaRa
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcdna il 4 - by Bioz Stars, 2020-09
    86/100 stars
      Buy from Supplier

    88
    TaKaRa pcdna fpn enhanced green fluorescent protein egfp construct
    Alteration of miR-20a or <t>FPN</t> expression affects NSCLC proliferation. H1299 cells were transiently transfected with either miR-20a inhibitor, a negative control (NC), the expression plasmid <t>pcDNA</t> FPN-flag, or a control vector (pcDNA empty). H1650 cells were transiently transfected with either miR-20a mimic, a negative control (NC), specific FPN siRNAs (FPN siRNA 1 and FPN siRNA 2) or NC siRNA. a FPN mRNA levels analyzed by qPCR in H1650 cells. qPCR data were normalized to ACTB. **** P
    Pcdna Fpn Enhanced Green Fluorescent Protein Egfp Construct, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna fpn enhanced green fluorescent protein egfp construct/product/TaKaRa
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcdna fpn enhanced green fluorescent protein egfp construct - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    85
    TaKaRa pcdna mgc2663
    Immunoprecipitation of transfected CV-1 cells to demonstrate that <t>MGC2663</t> associates with RTA in vivo. CV-1 cells were cotransfected with <t>pcDNA-ORF50</t> and pHA-MGC2663 (lanes 1 and 2) or with pHA-MGC2663 alone (lane 3). The cell lysates were precipitated with RTA antiserum (lanes 2 and 3), and the coimmunoprecipitation of MGC2663 was detected using anti-HA antibody. Expression of HA-tagged MGC2663 in the total lysates was also indicated in the same Western blot as a control (lane 1).
    Pcdna Mgc2663, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna mgc2663/product/TaKaRa
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    pcdna mgc2663 - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    92
    TaKaRa trbp dddd pcdna 3 1
    The phosphorylated <t>TRBP</t> isoform efficiently reverses PKR’s growth inhibition phenotype in yeast. ( A ) Yeast growth inhibition assay. Yeast INVSc1 cells were co-transformed with wt PKR/pYES2 and empty vector pYES3CT (PKR alone), wt PKR/pYES2 and K296R PKR/pYES3CT (PKR+K296R), wt PKR/pYES2 and wt TRBP/pYES3CT (PKR+wtTRBP), wt PKR/pYES2 and AAAA TRBP/pYES3CT (PKR+AAAA), or wt PKR/pYES2 and <t>DDDD</t> TRBP/pYES3CT (PKR + DDDD). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on double dropout media (-uracil, - tryptophan) with either glucose (+GLU) or galactose (+GAL) as sole carbon source. Plates were incubated for three days at 30 °C. Transformation of INVSc1 with wt PKR/pYES2 and empty vector pYES3CT served as a control showing growth inhibition on galactose plates, while transformation with wtPKR/pYES2 and K296R PKR/pYES3CT served as a positive control for inhibition of PKR and a reversal of growth inhibition phenotype.
    Trbp Dddd Pcdna 3 1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trbp dddd pcdna 3 1/product/TaKaRa
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    trbp dddd pcdna 3 1 - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    RT-PCR results for the transfected cells. M, DL2000 DNA marker; Lanes 1, 3, and 5, RT-PCR products from cells transfected with pcDNA3.1(+); Lane 2, RT-PCR product from cells transfected with pcDNA-IL-2; Lane 4, RT-PCR product from cells transfected with pcDNA-IL-4; Lane 6, RT-PCR product from cells transfected with pcDNA-ORF5.

    Journal: Journal of Veterinary Science

    Article Title: Positive effects of porcine IL-2 and IL-4 on virus-specific immune responses induced by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 DNA vaccine in swine

    doi: 10.4142/jvs.2014.15.1.99

    Figure Lengend Snippet: RT-PCR results for the transfected cells. M, DL2000 DNA marker; Lanes 1, 3, and 5, RT-PCR products from cells transfected with pcDNA3.1(+); Lane 2, RT-PCR product from cells transfected with pcDNA-IL-2; Lane 4, RT-PCR product from cells transfected with pcDNA-IL-4; Lane 6, RT-PCR product from cells transfected with pcDNA-ORF5.

    Article Snippet: Small-scale preparation of pcDNA-IL-2, pcDNA-IL-4, and pcDNA-ORF5 plasmids The E. coli Top10 cells which contained the recombinant plasmid pcDNA3.1(+)-ORF5 were used to inoculate 5 mL 2×YT liquid medium (Takara Bio, Japan) containing 50 µg/mL ampicillin, and incubated with shaking at 0.80 × g (120 rpm, rotational radius is 5 cm) at 37℃ for 12~16 h. Plasmid DNA was extracted with an E.Z.N.A.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Marker

    Detection of recombinant proteins encoded by expression plasmids pcDNA-ORF5(A), pcDNA-IL-2 (B), and pcDNA-IL-4 (C) using Western blotting.

    Journal: Journal of Veterinary Science

    Article Title: Positive effects of porcine IL-2 and IL-4 on virus-specific immune responses induced by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 DNA vaccine in swine

    doi: 10.4142/jvs.2014.15.1.99

    Figure Lengend Snippet: Detection of recombinant proteins encoded by expression plasmids pcDNA-ORF5(A), pcDNA-IL-2 (B), and pcDNA-IL-4 (C) using Western blotting.

    Article Snippet: Small-scale preparation of pcDNA-IL-2, pcDNA-IL-4, and pcDNA-ORF5 plasmids The E. coli Top10 cells which contained the recombinant plasmid pcDNA3.1(+)-ORF5 were used to inoculate 5 mL 2×YT liquid medium (Takara Bio, Japan) containing 50 µg/mL ampicillin, and incubated with shaking at 0.80 × g (120 rpm, rotational radius is 5 cm) at 37℃ for 12~16 h. Plasmid DNA was extracted with an E.Z.N.A.

    Techniques: Recombinant, Expressing, Western Blot

    Alteration of miR-20a or FPN expression affects NSCLC proliferation. H1299 cells were transiently transfected with either miR-20a inhibitor, a negative control (NC), the expression plasmid pcDNA FPN-flag, or a control vector (pcDNA empty). H1650 cells were transiently transfected with either miR-20a mimic, a negative control (NC), specific FPN siRNAs (FPN siRNA 1 and FPN siRNA 2) or NC siRNA. a FPN mRNA levels analyzed by qPCR in H1650 cells. qPCR data were normalized to ACTB. **** P

    Journal: Journal of Molecular Medicine (Berlin, Germany)

    Article Title: miR-20a regulates expression of the iron exporter ferroportin in lung cancer

    doi: 10.1007/s00109-015-1362-3

    Figure Lengend Snippet: Alteration of miR-20a or FPN expression affects NSCLC proliferation. H1299 cells were transiently transfected with either miR-20a inhibitor, a negative control (NC), the expression plasmid pcDNA FPN-flag, or a control vector (pcDNA empty). H1650 cells were transiently transfected with either miR-20a mimic, a negative control (NC), specific FPN siRNAs (FPN siRNA 1 and FPN siRNA 2) or NC siRNA. a FPN mRNA levels analyzed by qPCR in H1650 cells. qPCR data were normalized to ACTB. **** P

    Article Snippet: Plasmid cloning and site-directed mutagenesis To generate the pcDNA FPN-enhanced green fluorescent protein (EGFP) construct, the complete human FPN coding sequence was amplified from cDNA of HEK293 cells and inserted into the NheI-XheI restriction sites of the pEGFP-N1 vector (Clontech).

    Techniques: Expressing, Transfection, Negative Control, Plasmid Preparation, Real-time Polymerase Chain Reaction

    Colony formation of H1299 cells is affected by expression levels of miR-20a and FPN. H1299 cells were transiently transfected with either a miR-20a mimic, a miR-20 inhibitor, pcDNA FPN-flag, or FPN siRNA. Twenty-four hours later, the cells were seeded at a clonal density of 1000 cells/well in a 6-well plate. After 15 days of incubation, the colonies were counted using the Cell Counter v.2.1. Quantitative data are shown in a and b . Treatments are indicated. Left , representative pictures of colonies formed; right , quantitation of colonies. Experiments were performed in triplicates and repeated at least three times. Data were presented as mean ± SEM. * P

    Journal: Journal of Molecular Medicine (Berlin, Germany)

    Article Title: miR-20a regulates expression of the iron exporter ferroportin in lung cancer

    doi: 10.1007/s00109-015-1362-3

    Figure Lengend Snippet: Colony formation of H1299 cells is affected by expression levels of miR-20a and FPN. H1299 cells were transiently transfected with either a miR-20a mimic, a miR-20 inhibitor, pcDNA FPN-flag, or FPN siRNA. Twenty-four hours later, the cells were seeded at a clonal density of 1000 cells/well in a 6-well plate. After 15 days of incubation, the colonies were counted using the Cell Counter v.2.1. Quantitative data are shown in a and b . Treatments are indicated. Left , representative pictures of colonies formed; right , quantitation of colonies. Experiments were performed in triplicates and repeated at least three times. Data were presented as mean ± SEM. * P

    Article Snippet: Plasmid cloning and site-directed mutagenesis To generate the pcDNA FPN-enhanced green fluorescent protein (EGFP) construct, the complete human FPN coding sequence was amplified from cDNA of HEK293 cells and inserted into the NheI-XheI restriction sites of the pEGFP-N1 vector (Clontech).

    Techniques: Expressing, Transfection, Incubation, Quantitation Assay

    Immunoprecipitation of transfected CV-1 cells to demonstrate that MGC2663 associates with RTA in vivo. CV-1 cells were cotransfected with pcDNA-ORF50 and pHA-MGC2663 (lanes 1 and 2) or with pHA-MGC2663 alone (lane 3). The cell lysates were precipitated with RTA antiserum (lanes 2 and 3), and the coimmunoprecipitation of MGC2663 was detected using anti-HA antibody. Expression of HA-tagged MGC2663 in the total lysates was also indicated in the same Western blot as a control (lane 1).

    Journal: Journal of Virology

    Article Title: Identification of a Cellular Protein That Interacts and Synergizes with the RTA (ORF50) Protein of Kaposi's Sarcoma-Associated Herpesvirus in Transcriptional Activation

    doi: 10.1128/JVI.75.24.11961-11973.2001

    Figure Lengend Snippet: Immunoprecipitation of transfected CV-1 cells to demonstrate that MGC2663 associates with RTA in vivo. CV-1 cells were cotransfected with pcDNA-ORF50 and pHA-MGC2663 (lanes 1 and 2) or with pHA-MGC2663 alone (lane 3). The cell lysates were precipitated with RTA antiserum (lanes 2 and 3), and the coimmunoprecipitation of MGC2663 was detected using anti-HA antibody. Expression of HA-tagged MGC2663 in the total lysates was also indicated in the same Western blot as a control (lane 1).

    Article Snippet: Plasmid pEGFP-MGC2663, which expresses MGC2663-green fluorescent protein (GFP) fusion protein, was generated by inserting the MGC2663 Xho I/ Eco RI fragment of pcDNA-MGC2663 into the corresponding sites of the pEGFP-N1 vector (Clontech).

    Techniques: Immunoprecipitation, Transfection, In Vivo, Expressing, Western Blot

    Northern blot analysis of the expression pattern of MGC2663 gene in various mammalian cell lines. (A) MGC2663 transcript was detected in all human cells tested and in CV-1 cells. Total cellular RNAs were extracted from BC-3 cells (untreated [BC-3] or TPA treated for 24 h [BC-3/TPA]) and BJAB cells (transfected with vector control [BJAB] or pcDNA-MGC2663 [BJAB/MGC2663]), as well as transfected (/MGC2663) or control 293 cells, 3T3 cells, and CV-1 cells. Northern blotting was performed using a 32 P-labeled MGC2663-specific probe and analyzed by a PhosphorImager after a 2-day exposure. The RNA sizes according to the molecular size markers are indicated. The same membrane was then rehybridized with a 32 P-labeled GAPDH probe to ensure similar amounts of RNA were loaded onto each lane. (B) Northern blot analysis of the RTA RNA in BC-3 cells. The same membrane used for panel A was stripped and reprobed with a 32 P-labeled ORF50 probe. A 3.6-kb RTA band was detected in both control and TPA-stimulated BC-3 cells.

    Journal: Journal of Virology

    Article Title: Identification of a Cellular Protein That Interacts and Synergizes with the RTA (ORF50) Protein of Kaposi's Sarcoma-Associated Herpesvirus in Transcriptional Activation

    doi: 10.1128/JVI.75.24.11961-11973.2001

    Figure Lengend Snippet: Northern blot analysis of the expression pattern of MGC2663 gene in various mammalian cell lines. (A) MGC2663 transcript was detected in all human cells tested and in CV-1 cells. Total cellular RNAs were extracted from BC-3 cells (untreated [BC-3] or TPA treated for 24 h [BC-3/TPA]) and BJAB cells (transfected with vector control [BJAB] or pcDNA-MGC2663 [BJAB/MGC2663]), as well as transfected (/MGC2663) or control 293 cells, 3T3 cells, and CV-1 cells. Northern blotting was performed using a 32 P-labeled MGC2663-specific probe and analyzed by a PhosphorImager after a 2-day exposure. The RNA sizes according to the molecular size markers are indicated. The same membrane was then rehybridized with a 32 P-labeled GAPDH probe to ensure similar amounts of RNA were loaded onto each lane. (B) Northern blot analysis of the RTA RNA in BC-3 cells. The same membrane used for panel A was stripped and reprobed with a 32 P-labeled ORF50 probe. A 3.6-kb RTA band was detected in both control and TPA-stimulated BC-3 cells.

    Article Snippet: Plasmid pEGFP-MGC2663, which expresses MGC2663-green fluorescent protein (GFP) fusion protein, was generated by inserting the MGC2663 Xho I/ Eco RI fragment of pcDNA-MGC2663 into the corresponding sites of the pEGFP-N1 vector (Clontech).

    Techniques: Northern Blot, Expressing, Transfection, Plasmid Preparation, Labeling

    Enhancement of RTA transactivation of various KSHV promoters by MGC2663. (A) Transfection of CV-1 cells was carried out with 50 ng of viral promoter reporter construct and 250 ng of pcDNA-ORF50 (RTA) and/or 500 ng of pcDNA-MGC2663 (MGC2663) (+) as indicated. The total DNA amount used in each transfection was normalized by adding pcDNA3.1(−) vector. Luciferase activities were measured 48 h posttransfection. The error bars indicate standard deviations. (B and C) Transfections of 3T3 and 293 cells were carried out as described for panel A, using the same amounts of DNA for transfection. (D) Transfection of BJAB cells. In each transfection, 10 7 BJAB cells were mixed with 2 μg of reporter plasmid, 5 μg of RTA expression plasmid, and/or 5 μg of MGC2663 expression plasmid. Transfection was carried out using electroporation as described in Materials and Methods. (E) MGC2663 stimulation of RTA activation is dose dependent and is specific for RTA and its target promoters. CV-1 cells were transfected with 50 ng of KSHV promoter reporter construct, pGL3-promoter, or pHIVLTR-luc, as well as the indicated amount of pcDNA-ORF50, pcDNA-Tat, and/or pcDNA-MGC2663. Each result represents an average of at least three independent experiments. The standard deviations are shown as error bars. Transfection efficiency for each experiment was normalized using a β-Gal expression plasmid as an internal control. Fold activation was calculated based on the transfection of the reporter plasmid and the vector control, which was normalized to 1. (F) Western blot analysis of RTA expression in transfected CV-1 cells in the presence or absence of MGC2663. Lysates of cells transfected with either pcDNA-ORF50 alone (lane 1) or pcDNA-ORF50 and pcDNA-MGC2663 (lane 2) were analyzed using RTA antiserum. The protein molecular mass markers are indicated.

    Journal: Journal of Virology

    Article Title: Identification of a Cellular Protein That Interacts and Synergizes with the RTA (ORF50) Protein of Kaposi's Sarcoma-Associated Herpesvirus in Transcriptional Activation

    doi: 10.1128/JVI.75.24.11961-11973.2001

    Figure Lengend Snippet: Enhancement of RTA transactivation of various KSHV promoters by MGC2663. (A) Transfection of CV-1 cells was carried out with 50 ng of viral promoter reporter construct and 250 ng of pcDNA-ORF50 (RTA) and/or 500 ng of pcDNA-MGC2663 (MGC2663) (+) as indicated. The total DNA amount used in each transfection was normalized by adding pcDNA3.1(−) vector. Luciferase activities were measured 48 h posttransfection. The error bars indicate standard deviations. (B and C) Transfections of 3T3 and 293 cells were carried out as described for panel A, using the same amounts of DNA for transfection. (D) Transfection of BJAB cells. In each transfection, 10 7 BJAB cells were mixed with 2 μg of reporter plasmid, 5 μg of RTA expression plasmid, and/or 5 μg of MGC2663 expression plasmid. Transfection was carried out using electroporation as described in Materials and Methods. (E) MGC2663 stimulation of RTA activation is dose dependent and is specific for RTA and its target promoters. CV-1 cells were transfected with 50 ng of KSHV promoter reporter construct, pGL3-promoter, or pHIVLTR-luc, as well as the indicated amount of pcDNA-ORF50, pcDNA-Tat, and/or pcDNA-MGC2663. Each result represents an average of at least three independent experiments. The standard deviations are shown as error bars. Transfection efficiency for each experiment was normalized using a β-Gal expression plasmid as an internal control. Fold activation was calculated based on the transfection of the reporter plasmid and the vector control, which was normalized to 1. (F) Western blot analysis of RTA expression in transfected CV-1 cells in the presence or absence of MGC2663. Lysates of cells transfected with either pcDNA-ORF50 alone (lane 1) or pcDNA-ORF50 and pcDNA-MGC2663 (lane 2) were analyzed using RTA antiserum. The protein molecular mass markers are indicated.

    Article Snippet: Plasmid pEGFP-MGC2663, which expresses MGC2663-green fluorescent protein (GFP) fusion protein, was generated by inserting the MGC2663 Xho I/ Eco RI fragment of pcDNA-MGC2663 into the corresponding sites of the pEGFP-N1 vector (Clontech).

    Techniques: Transfection, Construct, Plasmid Preparation, Luciferase, Expressing, Electroporation, Activation Assay, Western Blot

    The phosphorylated TRBP isoform efficiently reverses PKR’s growth inhibition phenotype in yeast. ( A ) Yeast growth inhibition assay. Yeast INVSc1 cells were co-transformed with wt PKR/pYES2 and empty vector pYES3CT (PKR alone), wt PKR/pYES2 and K296R PKR/pYES3CT (PKR+K296R), wt PKR/pYES2 and wt TRBP/pYES3CT (PKR+wtTRBP), wt PKR/pYES2 and AAAA TRBP/pYES3CT (PKR+AAAA), or wt PKR/pYES2 and DDDD TRBP/pYES3CT (PKR + DDDD). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on double dropout media (-uracil, - tryptophan) with either glucose (+GLU) or galactose (+GAL) as sole carbon source. Plates were incubated for three days at 30 °C. Transformation of INVSc1 with wt PKR/pYES2 and empty vector pYES3CT served as a control showing growth inhibition on galactose plates, while transformation with wtPKR/pYES2 and K296R PKR/pYES3CT served as a positive control for inhibition of PKR and a reversal of growth inhibition phenotype.

    Journal: Scientific Reports

    Article Title: Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival

    doi: 10.1038/s41598-018-19360-8

    Figure Lengend Snippet: The phosphorylated TRBP isoform efficiently reverses PKR’s growth inhibition phenotype in yeast. ( A ) Yeast growth inhibition assay. Yeast INVSc1 cells were co-transformed with wt PKR/pYES2 and empty vector pYES3CT (PKR alone), wt PKR/pYES2 and K296R PKR/pYES3CT (PKR+K296R), wt PKR/pYES2 and wt TRBP/pYES3CT (PKR+wtTRBP), wt PKR/pYES2 and AAAA TRBP/pYES3CT (PKR+AAAA), or wt PKR/pYES2 and DDDD TRBP/pYES3CT (PKR + DDDD). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on double dropout media (-uracil, - tryptophan) with either glucose (+GLU) or galactose (+GAL) as sole carbon source. Plates were incubated for three days at 30 °C. Transformation of INVSc1 with wt PKR/pYES2 and empty vector pYES3CT served as a control showing growth inhibition on galactose plates, while transformation with wtPKR/pYES2 and K296R PKR/pYES3CT served as a positive control for inhibition of PKR and a reversal of growth inhibition phenotype.

    Article Snippet: HeLa cells were grown to 50% confluency in six-well plates and co-transfected with 200 ng of Flag wt TRBP, TRBP AAAA or TRBP DDDD/pcDNA 3.1− and 200 ng of pEGFPC1 (Clontech) using Effectene (Qiagen).

    Techniques: Inhibition, Growth Inhibition Assay, Transformation Assay, Plasmid Preparation, Incubation, Positive Control

    TRBP phosphorylation inhibits PKR-mediated apoptosis during cell stress. ( A ) Schematic representation of TRBP phosphorylation sites. Blue boxes represent the three double-stranded RNA binding motifs (dsRBMs), M1, M2, and M3. Red vertical lines represent previously identified ERK 1/2 phosphorylation sites at S142, S152, S283, and S286. ( B ) Expression of phospho-mimic TRBP protects cells during oxidative stress. HeLa cells were transfected with 200 ng pEGFPC1 (EV) alone (black bars) or with 200 ng each of pEGFPC1 and Flag TRBP AAAA/pcDNA 3.1 − (blue bars), 200 ng each of pEGFPC1 and Flag TRBP DDDD/pcDNA 3.1 − (red bars) or 200 ng each of pEGFPC1 and Flag wt TRBP/pcDNA 3.1 − (green bars). 24 hours after transfection, the cells were treated with 25 μM sodium arsenite, fixed and stained with DAPI nuclear stain. At least 300 EGFP-positive cells were scored as apoptotic or live based on nuclear condensation indicated by intense DAPI nuclear staining and cell morphology. The percentage of cells undergoing apoptosis (% apoptosis) was calculated using the formula: (EGFP- expressing cells with intense DAPI nuclear staining/Total EGFP-expressing cells) x 100. Bars represent averages ± S.D. from three independent experiments. One-way ANOVA followed by post-hoc Tukey test was performed, ns: not significant, asterisk *p value of 0.043. asterisk **p value of 0.007. ( C ) and ( D ) Phospho-mimic TRBP inhibits PKR mediated apoptosis more efficiently than phospho-defective TRBP ( C ) HeLa cells were plated on coverslips and transfected with 500 ng of wt PKR/pEGFPC1 and 20 ng of empty vector pCDNA3.1 − (wt PKR; black bar) or with 500 ng of wt PKR/pEGFPC1 + 20 ng of Flag TRBP AAAA/pcDNA 3.1 − (blue bar) or 500 ng of wt PKR/pEGFPC1 + 20 ng of Flag TRBP DDDD/ pcDNA 3.1 − (red bar). 24 hours after transfection, the cells were fixed and mounted in Vectashield mounting media with DAPI nuclear stain. Representative fluorescent micrographs of HeLa cells transfected with wt PKR pEGFPC1 alone (Panel A), or in combination with Flag TRBP AAAA/pcDNA 3.1 − (Panel B) or Flag TRBP DDDD/pcDNA 3.1 − (Panel C) are shown. At least 500 EGFP-PKR expressing cells showing green fluorescence were scored as apoptotic (white arrows) or live (white arrowheads) based on nuclear condensation indicated by intense DAPI staining and cellular morphology. The cells showing intense DAPI staining and rounded morphology were scored as apoptotic. The percentage of cells undergoing apoptosis was determined as described in ( A ). Bars represent averages ± S.D. from three independent experiments. One-way ANOVA followed by post-hoc Tukey test was performed, asterisk *p value 0.0034, double asterisk **p value 0.0002, # p value 0.0134. ( D ) Phospho-mimic TRBP expression abrogates mitochondrial depolarization during PKR-mediated apoptosis. HeLa cells were plated on coverslips and transfected with 500 ng of wt PKR/pEGFPC1 and 20 ng of empty vector pCDNA3.1 − (wt PKR; black bar) or with 500 ng of wt PKR/pEGFPC1 + 20 ng of Flag TRBP AAAA/pcDNA 3.1 − (blue bar) or 500 ng of wt PKR/pEGFPC1 + 20 ng of Flag TRBP DDDD/ pcDNA 3.1 − (red bar). 24 hours after transfection, changes in the mitochondrial potential of transfected cells were assessed using the MitoPT TMRM Assay kit and observed by fluorescence microscopy. Representative fluorescent micrographs of the cells transfected with wt-PKR pEGFPC1 alone (wt-PKR-EGFP + EV, Panel A), and AAAA TRBP (wt-PKR-EGFP + AAAA, Panel B) or DDDD TRBP (wt-PKR-EGFP + DDDD, Panel C) are represented. At least 500 PKR expressing cells (GFP positive, green fluorescent cells) were scored as live (white arrowheads) or dead (white arrows) based on decreased or absent red fluorescence. The PKR expressing, green fluorescent cells that showed diminished or no MitoPT staining (red fluorescence) were counted as apoptotic and the green fluorescent cells that showed good MitoPT staining (red fluorescence) were counted as live. The percentage of cells undergoing apoptosis (% apoptosis) was calculated using the formula: (EGFP- expressing cells with decreased or absent red fluorescence/Total EGFP-expressing cells) x 100. Bars represent averages ± S.D. from three independent experiments. One-way ANOVA followed by post-hoc Tukey test was performed, asterisk *p value 0.0108, double asterisk **p value 0.0003, # p value 0.0002.

    Journal: Scientific Reports

    Article Title: Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival

    doi: 10.1038/s41598-018-19360-8

    Figure Lengend Snippet: TRBP phosphorylation inhibits PKR-mediated apoptosis during cell stress. ( A ) Schematic representation of TRBP phosphorylation sites. Blue boxes represent the three double-stranded RNA binding motifs (dsRBMs), M1, M2, and M3. Red vertical lines represent previously identified ERK 1/2 phosphorylation sites at S142, S152, S283, and S286. ( B ) Expression of phospho-mimic TRBP protects cells during oxidative stress. HeLa cells were transfected with 200 ng pEGFPC1 (EV) alone (black bars) or with 200 ng each of pEGFPC1 and Flag TRBP AAAA/pcDNA 3.1 − (blue bars), 200 ng each of pEGFPC1 and Flag TRBP DDDD/pcDNA 3.1 − (red bars) or 200 ng each of pEGFPC1 and Flag wt TRBP/pcDNA 3.1 − (green bars). 24 hours after transfection, the cells were treated with 25 μM sodium arsenite, fixed and stained with DAPI nuclear stain. At least 300 EGFP-positive cells were scored as apoptotic or live based on nuclear condensation indicated by intense DAPI nuclear staining and cell morphology. The percentage of cells undergoing apoptosis (% apoptosis) was calculated using the formula: (EGFP- expressing cells with intense DAPI nuclear staining/Total EGFP-expressing cells) x 100. Bars represent averages ± S.D. from three independent experiments. One-way ANOVA followed by post-hoc Tukey test was performed, ns: not significant, asterisk *p value of 0.043. asterisk **p value of 0.007. ( C ) and ( D ) Phospho-mimic TRBP inhibits PKR mediated apoptosis more efficiently than phospho-defective TRBP ( C ) HeLa cells were plated on coverslips and transfected with 500 ng of wt PKR/pEGFPC1 and 20 ng of empty vector pCDNA3.1 − (wt PKR; black bar) or with 500 ng of wt PKR/pEGFPC1 + 20 ng of Flag TRBP AAAA/pcDNA 3.1 − (blue bar) or 500 ng of wt PKR/pEGFPC1 + 20 ng of Flag TRBP DDDD/ pcDNA 3.1 − (red bar). 24 hours after transfection, the cells were fixed and mounted in Vectashield mounting media with DAPI nuclear stain. Representative fluorescent micrographs of HeLa cells transfected with wt PKR pEGFPC1 alone (Panel A), or in combination with Flag TRBP AAAA/pcDNA 3.1 − (Panel B) or Flag TRBP DDDD/pcDNA 3.1 − (Panel C) are shown. At least 500 EGFP-PKR expressing cells showing green fluorescence were scored as apoptotic (white arrows) or live (white arrowheads) based on nuclear condensation indicated by intense DAPI staining and cellular morphology. The cells showing intense DAPI staining and rounded morphology were scored as apoptotic. The percentage of cells undergoing apoptosis was determined as described in ( A ). Bars represent averages ± S.D. from three independent experiments. One-way ANOVA followed by post-hoc Tukey test was performed, asterisk *p value 0.0034, double asterisk **p value 0.0002, # p value 0.0134. ( D ) Phospho-mimic TRBP expression abrogates mitochondrial depolarization during PKR-mediated apoptosis. HeLa cells were plated on coverslips and transfected with 500 ng of wt PKR/pEGFPC1 and 20 ng of empty vector pCDNA3.1 − (wt PKR; black bar) or with 500 ng of wt PKR/pEGFPC1 + 20 ng of Flag TRBP AAAA/pcDNA 3.1 − (blue bar) or 500 ng of wt PKR/pEGFPC1 + 20 ng of Flag TRBP DDDD/ pcDNA 3.1 − (red bar). 24 hours after transfection, changes in the mitochondrial potential of transfected cells were assessed using the MitoPT TMRM Assay kit and observed by fluorescence microscopy. Representative fluorescent micrographs of the cells transfected with wt-PKR pEGFPC1 alone (wt-PKR-EGFP + EV, Panel A), and AAAA TRBP (wt-PKR-EGFP + AAAA, Panel B) or DDDD TRBP (wt-PKR-EGFP + DDDD, Panel C) are represented. At least 500 PKR expressing cells (GFP positive, green fluorescent cells) were scored as live (white arrowheads) or dead (white arrows) based on decreased or absent red fluorescence. The PKR expressing, green fluorescent cells that showed diminished or no MitoPT staining (red fluorescence) were counted as apoptotic and the green fluorescent cells that showed good MitoPT staining (red fluorescence) were counted as live. The percentage of cells undergoing apoptosis (% apoptosis) was calculated using the formula: (EGFP- expressing cells with decreased or absent red fluorescence/Total EGFP-expressing cells) x 100. Bars represent averages ± S.D. from three independent experiments. One-way ANOVA followed by post-hoc Tukey test was performed, asterisk *p value 0.0108, double asterisk **p value 0.0003, # p value 0.0002.

    Article Snippet: HeLa cells were grown to 50% confluency in six-well plates and co-transfected with 200 ng of Flag wt TRBP, TRBP AAAA or TRBP DDDD/pcDNA 3.1− and 200 ng of pEGFPC1 (Clontech) using Effectene (Qiagen).

    Techniques: RNA Binding Assay, Expressing, Transfection, Staining, Plasmid Preparation, Fluorescence, Microscopy

    TRBP phosphorylation strengthens PKR-TRBP interaction and weakens TRBP-TRBP interaction. ( A ) Phospho-mimic TRBP mutant interacts stronger with PKR compared to the phospho-defective TRBP mutant in yeast two-hybrid assay. PKR/pGAD424 and either AAAA TRBP/pGBKT7, DDDD TRBP/pGBKT7, or wt TRBP/pGBKT7 were co-transformed into AH109 yeast cells and selected on SD double dropout media (-tryptophan, - leucine). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on SD triple dropout media (-tryptophan, - leucine, - histidine) containing 10 mM 3-amino-1,2,4-triazole (3-AT). Plates were incubated for 3 days at 30 °C. Transformation of PKR in pGAD424 and pGBKT7 empty vector served as a negative control. ( B ) Phosphomimic TRBP mutant shows stronger heteromeric interaction with PKR compared to the phosphodefective TRBP mutant in mammalian cells. HeLa cells were transfected with Flag K296R PKR/pcDNA 3.1 − and either myc TRBP AAAA/pcDNA 3.1 − , myc wt TRBP/pcDNA 3.1 − , or myc TRBP DDDD/pcDNA 3.1 − . The cells were harvested 24 hours after transfection, and myc AAAA, DDDD or wt TRBP was immunoprecipitated using anti-myc monoclonal antibody conjugated agarose beads. Co-immunoprecipitated Flag PKR was analyzed by western blot analysis with an anti-Flag antibody (IP: x Flag (PKR) panel). The blot was subsequently re-probed with anti-myc antibody to ensure equal myc TRBP immunoprecipitation from each sample (IP: x myc (TRBP) panel). Equal Flag PKR and myc TRBP expression in all samples was tested by western blot analysis of equal amounts of total cell lysate with anti-myc, and anti-Flag antibodies (input: x Flag (PKR) and x myc (TRBP) panels). ( C ) Changes in TRBP association with PKR. Flag TRBP overexpressing cells were treated with 25 μM sodium arsenite for the indicated time points. Cell extracts were prepared in the presence of a phosphatase inhibitor and 25 μg of cell extract was incubated with 500 ng of pure recombinant hexahistidine (His)-tagged PKR immobilized on Ni 2+ -agarose beads. After washing the beads, PKR-associated Flag TRBP was analyzed by SDS polyacrylamide gel electrophoresis followed by western blot analysis with anti-Flag antibody. Western blot analysis was also performed with anti-His antibody to ensure equal His- PKR in each sample. 25 μg of cell extract was also analyzed by western blot analysis with anti-Flag and anti-GAPDH antibodies to ensure equal addition of cell lysate for each pull down (Input). Quantification of TRBP-PKR pull down: Band intensities were quantified using ImageQuant TL Software, and the ratios of bound TRBP to bound PKR across all samples were calculated and normalized to the band intensities of Flag-TRBP input for each sample. Bound TRBP/his-PKR ratios for all samples were all expressed relative to the control sample (Lane 2). Averages from three independent experiments are plotted as bar graphs ± S.D. One-way ANOVA followed by post-hoc Tukey test was performed, asterisk *p value 0.0000012 and double asterisk **p value 0.0066374. ( D ) Phosphomimic TRBP mutant shows stronger homomeric interaction compared to the phosphodefective TRBP mutant in yeast two-hybrid assay. AAAA TRBP or DDDD TRBP point mutants in pGADT7 and pGBKT7 were co-transformed into AH109 yeast cells and selected on SD double dropout media (-tryptophan, -leucine). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on SD triple dropout media plate (tryptophan, -leucine, -histidine) containing 10 mM 3-amino-1,2,4-triazole (3-AT). Plates were incubated for 5 days at 30 °C. Transformation of pGADT7 and pGBKT7 empty vectors served as a negative control. ( E ) Phosphomimic TRBP mutant shows stronger homomeric interaction compared to the phosphodefective TRBP mutant in mammalian cells. HeLa cells were transfected with either myc TRBP DDDD/pcDNA 3.1 − and Flag TRBP DDDD/pcDNA 3.1 − or Flag TRBP AAAA/pcDNA 3.1 − and myc TRBP AAAA/pcDNA 3.1 − . The cells were harvested 24 hours after transfection, and Flag TRBP AAAA or DDDD was immunoprecipitated using anti-Flag monoclonal antibody conjugated agarose beads. The co-immunoprecipitation of myc-TRBP was analyzed by western blot analysis with an anti-myc antibody (IP: x Myc panel). Blot was subsequently stripped and re-probed with anti-Flag antibody to ensure equal Flag-TRBP immunoprecipitation from each sample (IP: x Flag panel). Equal AAAA TRBP and DDDD TRBP expression in all samples was tested by western blot analysis of equal amounts of total cell lysate with anti-myc, and anti-Flag antibodies (Input: x Myc and x Flag panels).

    Journal: Scientific Reports

    Article Title: Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival

    doi: 10.1038/s41598-018-19360-8

    Figure Lengend Snippet: TRBP phosphorylation strengthens PKR-TRBP interaction and weakens TRBP-TRBP interaction. ( A ) Phospho-mimic TRBP mutant interacts stronger with PKR compared to the phospho-defective TRBP mutant in yeast two-hybrid assay. PKR/pGAD424 and either AAAA TRBP/pGBKT7, DDDD TRBP/pGBKT7, or wt TRBP/pGBKT7 were co-transformed into AH109 yeast cells and selected on SD double dropout media (-tryptophan, - leucine). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on SD triple dropout media (-tryptophan, - leucine, - histidine) containing 10 mM 3-amino-1,2,4-triazole (3-AT). Plates were incubated for 3 days at 30 °C. Transformation of PKR in pGAD424 and pGBKT7 empty vector served as a negative control. ( B ) Phosphomimic TRBP mutant shows stronger heteromeric interaction with PKR compared to the phosphodefective TRBP mutant in mammalian cells. HeLa cells were transfected with Flag K296R PKR/pcDNA 3.1 − and either myc TRBP AAAA/pcDNA 3.1 − , myc wt TRBP/pcDNA 3.1 − , or myc TRBP DDDD/pcDNA 3.1 − . The cells were harvested 24 hours after transfection, and myc AAAA, DDDD or wt TRBP was immunoprecipitated using anti-myc monoclonal antibody conjugated agarose beads. Co-immunoprecipitated Flag PKR was analyzed by western blot analysis with an anti-Flag antibody (IP: x Flag (PKR) panel). The blot was subsequently re-probed with anti-myc antibody to ensure equal myc TRBP immunoprecipitation from each sample (IP: x myc (TRBP) panel). Equal Flag PKR and myc TRBP expression in all samples was tested by western blot analysis of equal amounts of total cell lysate with anti-myc, and anti-Flag antibodies (input: x Flag (PKR) and x myc (TRBP) panels). ( C ) Changes in TRBP association with PKR. Flag TRBP overexpressing cells were treated with 25 μM sodium arsenite for the indicated time points. Cell extracts were prepared in the presence of a phosphatase inhibitor and 25 μg of cell extract was incubated with 500 ng of pure recombinant hexahistidine (His)-tagged PKR immobilized on Ni 2+ -agarose beads. After washing the beads, PKR-associated Flag TRBP was analyzed by SDS polyacrylamide gel electrophoresis followed by western blot analysis with anti-Flag antibody. Western blot analysis was also performed with anti-His antibody to ensure equal His- PKR in each sample. 25 μg of cell extract was also analyzed by western blot analysis with anti-Flag and anti-GAPDH antibodies to ensure equal addition of cell lysate for each pull down (Input). Quantification of TRBP-PKR pull down: Band intensities were quantified using ImageQuant TL Software, and the ratios of bound TRBP to bound PKR across all samples were calculated and normalized to the band intensities of Flag-TRBP input for each sample. Bound TRBP/his-PKR ratios for all samples were all expressed relative to the control sample (Lane 2). Averages from three independent experiments are plotted as bar graphs ± S.D. One-way ANOVA followed by post-hoc Tukey test was performed, asterisk *p value 0.0000012 and double asterisk **p value 0.0066374. ( D ) Phosphomimic TRBP mutant shows stronger homomeric interaction compared to the phosphodefective TRBP mutant in yeast two-hybrid assay. AAAA TRBP or DDDD TRBP point mutants in pGADT7 and pGBKT7 were co-transformed into AH109 yeast cells and selected on SD double dropout media (-tryptophan, -leucine). Ten microliters of transformed yeast cells (OD 600 = 10, 1, 0.1, 0.01) were spotted on SD triple dropout media plate (tryptophan, -leucine, -histidine) containing 10 mM 3-amino-1,2,4-triazole (3-AT). Plates were incubated for 5 days at 30 °C. Transformation of pGADT7 and pGBKT7 empty vectors served as a negative control. ( E ) Phosphomimic TRBP mutant shows stronger homomeric interaction compared to the phosphodefective TRBP mutant in mammalian cells. HeLa cells were transfected with either myc TRBP DDDD/pcDNA 3.1 − and Flag TRBP DDDD/pcDNA 3.1 − or Flag TRBP AAAA/pcDNA 3.1 − and myc TRBP AAAA/pcDNA 3.1 − . The cells were harvested 24 hours after transfection, and Flag TRBP AAAA or DDDD was immunoprecipitated using anti-Flag monoclonal antibody conjugated agarose beads. The co-immunoprecipitation of myc-TRBP was analyzed by western blot analysis with an anti-myc antibody (IP: x Myc panel). Blot was subsequently stripped and re-probed with anti-Flag antibody to ensure equal Flag-TRBP immunoprecipitation from each sample (IP: x Flag panel). Equal AAAA TRBP and DDDD TRBP expression in all samples was tested by western blot analysis of equal amounts of total cell lysate with anti-myc, and anti-Flag antibodies (Input: x Myc and x Flag panels).

    Article Snippet: HeLa cells were grown to 50% confluency in six-well plates and co-transfected with 200 ng of Flag wt TRBP, TRBP AAAA or TRBP DDDD/pcDNA 3.1− and 200 ng of pEGFPC1 (Clontech) using Effectene (Qiagen).

    Techniques: Mutagenesis, Y2H Assay, Transformation Assay, Incubation, Plasmid Preparation, Negative Control, Transfection, Immunoprecipitation, Western Blot, Expressing, Recombinant, Polyacrylamide Gel Electrophoresis, Software