mammalian expression vector pcdna 3 1  (Thermo Fisher)


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    Structured Review

    Thermo Fisher mammalian expression vector pcdna 3 1
    Mammalian Expression Vector Pcdna 3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mammalian expression vector pcdna 3 1/product/Thermo Fisher
    Average 91 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    mammalian expression vector pcdna 3 1 - by Bioz Stars, 2020-04
    91/100 stars

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    Related Articles

    Clone Assay:

    Article Title: MicroRNA-23b attenuates the H2O2-induced injury of microglial cells via TAB3/NF-κB signaling pathway
    Article Snippet: .. The open reading frame (ORF) of human TAB3 without the 3’-UTR was generated by PCR amplification and then cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen; Life Technologies). .. Cell transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

    Transfection:

    Article Title: MicroRNA-23b attenuates the H2O2-induced injury of microglial cells via TAB3/NF-κB signaling pathway
    Article Snippet: Paragraph title: Transfection ... The open reading frame (ORF) of human TAB3 without the 3’-UTR was generated by PCR amplification and then cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen; Life Technologies).

    Amplification:

    Article Title: MicroRNA-23b attenuates the H2O2-induced injury of microglial cells via TAB3/NF-κB signaling pathway
    Article Snippet: .. The open reading frame (ORF) of human TAB3 without the 3’-UTR was generated by PCR amplification and then cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen; Life Technologies). .. Cell transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

    Synthesized:

    Article Title: MicroRNA-23b attenuates the H2O2-induced injury of microglial cells via TAB3/NF-κB signaling pathway
    Article Snippet: The miR-23b mimics, miR-23b inhibitor and negative control [ ] were synthesized by GenePharma (Shanghai, China). .. The open reading frame (ORF) of human TAB3 without the 3’-UTR was generated by PCR amplification and then cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen; Life Technologies).

    Negative Control:

    Article Title: MicroRNA-23b attenuates the H2O2-induced injury of microglial cells via TAB3/NF-κB signaling pathway
    Article Snippet: The miR-23b mimics, miR-23b inhibitor and negative control [ ] were synthesized by GenePharma (Shanghai, China). .. The open reading frame (ORF) of human TAB3 without the 3’-UTR was generated by PCR amplification and then cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen; Life Technologies).

    Generated:

    Article Title: MicroRNA-23b attenuates the H2O2-induced injury of microglial cells via TAB3/NF-κB signaling pathway
    Article Snippet: .. The open reading frame (ORF) of human TAB3 without the 3’-UTR was generated by PCR amplification and then cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen; Life Technologies). .. Cell transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

    Expressing:

    Article Title: MicroRNA-23b attenuates the H2O2-induced injury of microglial cells via TAB3/NF-κB signaling pathway
    Article Snippet: .. The open reading frame (ORF) of human TAB3 without the 3’-UTR was generated by PCR amplification and then cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen; Life Technologies). .. Cell transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

    Polymerase Chain Reaction:

    Article Title: MicroRNA-23b attenuates the H2O2-induced injury of microglial cells via TAB3/NF-κB signaling pathway
    Article Snippet: .. The open reading frame (ORF) of human TAB3 without the 3’-UTR was generated by PCR amplification and then cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen; Life Technologies). .. Cell transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

    Plasmid Preparation:

    Article Title: MicroRNA-23b attenuates the H2O2-induced injury of microglial cells via TAB3/NF-κB signaling pathway
    Article Snippet: .. The open reading frame (ORF) of human TAB3 without the 3’-UTR was generated by PCR amplification and then cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen; Life Technologies). .. Cell transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

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    Thermo Fisher pcdna3 1 vector
    Fgfr AS is regulated by Esrp genes in vertebrates and amphioxus. a RT-PCR assays showing differential Fgfr exon IIIb and IIIc inclusion in WT versus DMUT 5 d.p.f. zebrafish embryos. b RT-PCR assays for Fgfr AS in different amphioxus adult tissues, depicted in a transversal section. nc, nerve cord, ms, muscle, gl, gills, hd, hepatic diverticulum, nt, notochord, sk, skin. Reverse primers were designed in both exons IIIb and IIIc (arrows) and used together in the same PCR reaction. c Top: schematic representation of <t>pcDNA3.1-based</t> minigene constructs containing the genomic region spanning the Fgfr AS event of Branchiostoma lanceolatum , with (pcDNA3.1-BlaFGFR) and without (pcDNA3.1-BlaFGFRΔIIIx) exon IIIx. Bottom: relative intensity of fluorescent RT-PCR bands supporting differential inclusion of exons IIIb and IIIc when transfecting the minigenes alone (Control) or together with a plasmid containing either amphioxus or zebrafish full-length Esrp transcripts (BlaEsrp and DreEsrp1, respectively). Despite significant mis-splicing of the minigene in all conditions, only the amphioxus construct was able to induce a dramatic switch toward exon IIIb inclusion. Primers were designed in the neighboring constitutive exons (arrows). d RT-PCR assays for endogenous human AS events in the same control, BlaEsrp or DreEsrp1 transfected 293T cells showing that the amphioxus and zebrafish Esrp constructs are able to modulate endogenous Esrp -dependent events in a similar manner. Error bars correspond to standard errors of three biological replicates. Esrp -enhanced isoforms are marked with an isoform cartoon
    Pcdna3 1 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 605 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 vector/product/Thermo Fisher
    Average 99 stars, based on 605 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 vector - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher pcdna3 1
    SOCS1 physically interacts with RhTRIM5α. HEK293T cells were co-transfected with 1.0 µg of pRhTRIM5α-HA and 2.0 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 3.0 µg per sample with <t>pcDNA3.1.</t> Two days post-transfection, whole cell lysates were subjected to immunoprecipitation for RhTRIM5α with anti-HA antibody. Input lysates (left panels) and precipitated proteins (right panels) were separated by SDS-PAGE and proteins were detected by immunoblot analyses with anti-SOCS1 (upper panels) and anti-HA (RhTRIM5α-HA, lower panels) antibodies.
    Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 503 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1/product/Thermo Fisher
    Average 99 stars, based on 503 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    Fgfr AS is regulated by Esrp genes in vertebrates and amphioxus. a RT-PCR assays showing differential Fgfr exon IIIb and IIIc inclusion in WT versus DMUT 5 d.p.f. zebrafish embryos. b RT-PCR assays for Fgfr AS in different amphioxus adult tissues, depicted in a transversal section. nc, nerve cord, ms, muscle, gl, gills, hd, hepatic diverticulum, nt, notochord, sk, skin. Reverse primers were designed in both exons IIIb and IIIc (arrows) and used together in the same PCR reaction. c Top: schematic representation of pcDNA3.1-based minigene constructs containing the genomic region spanning the Fgfr AS event of Branchiostoma lanceolatum , with (pcDNA3.1-BlaFGFR) and without (pcDNA3.1-BlaFGFRΔIIIx) exon IIIx. Bottom: relative intensity of fluorescent RT-PCR bands supporting differential inclusion of exons IIIb and IIIc when transfecting the minigenes alone (Control) or together with a plasmid containing either amphioxus or zebrafish full-length Esrp transcripts (BlaEsrp and DreEsrp1, respectively). Despite significant mis-splicing of the minigene in all conditions, only the amphioxus construct was able to induce a dramatic switch toward exon IIIb inclusion. Primers were designed in the neighboring constitutive exons (arrows). d RT-PCR assays for endogenous human AS events in the same control, BlaEsrp or DreEsrp1 transfected 293T cells showing that the amphioxus and zebrafish Esrp constructs are able to modulate endogenous Esrp -dependent events in a similar manner. Error bars correspond to standard errors of three biological replicates. Esrp -enhanced isoforms are marked with an isoform cartoon

    Journal: Nature Communications

    Article Title: Evolutionary recruitment of flexible Esrp-dependent splicing programs into diverse embryonic morphogenetic processes

    doi: 10.1038/s41467-017-01961-y

    Figure Lengend Snippet: Fgfr AS is regulated by Esrp genes in vertebrates and amphioxus. a RT-PCR assays showing differential Fgfr exon IIIb and IIIc inclusion in WT versus DMUT 5 d.p.f. zebrafish embryos. b RT-PCR assays for Fgfr AS in different amphioxus adult tissues, depicted in a transversal section. nc, nerve cord, ms, muscle, gl, gills, hd, hepatic diverticulum, nt, notochord, sk, skin. Reverse primers were designed in both exons IIIb and IIIc (arrows) and used together in the same PCR reaction. c Top: schematic representation of pcDNA3.1-based minigene constructs containing the genomic region spanning the Fgfr AS event of Branchiostoma lanceolatum , with (pcDNA3.1-BlaFGFR) and without (pcDNA3.1-BlaFGFRΔIIIx) exon IIIx. Bottom: relative intensity of fluorescent RT-PCR bands supporting differential inclusion of exons IIIb and IIIc when transfecting the minigenes alone (Control) or together with a plasmid containing either amphioxus or zebrafish full-length Esrp transcripts (BlaEsrp and DreEsrp1, respectively). Despite significant mis-splicing of the minigene in all conditions, only the amphioxus construct was able to induce a dramatic switch toward exon IIIb inclusion. Primers were designed in the neighboring constitutive exons (arrows). d RT-PCR assays for endogenous human AS events in the same control, BlaEsrp or DreEsrp1 transfected 293T cells showing that the amphioxus and zebrafish Esrp constructs are able to modulate endogenous Esrp -dependent events in a similar manner. Error bars correspond to standard errors of three biological replicates. Esrp -enhanced isoforms are marked with an isoform cartoon

    Article Snippet: Amphioxus Fgfr AS minigenes and cell cultures The full-length Esrp open reading frame (ORF) from B. lanceolatum and esrp1 ORF from D. rerio were amplified from cDNA using iProof High Fidelity Polymerase (Bio-Rad) and cloned into the pcDNA3.1 vector (Thermo Fisher Scientific).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mass Spectrometry, Polymerase Chain Reaction, Construct, Plasmid Preparation, Transfection

    UCA1 acted as a ceRNA of miR129 to enhance target SOX4 gene expression in RCC cells. Notes: ( A ) 786O cells were transfected with miR129 mimic or miR-Con, and ACHN cells were transfected with miR129 inhibitor or anti-miR-Con, followed by the detection of SOX4 protein expression. ( B ) 786O cells were transfected with siCon or siUCA1-2, and ACHN cells were transfected with pcDNA3.1 vector or pcDNA-UCA1 plasmid, followed by the determination of SOX4 protein expression. ( C ) Effects of miR129 and UCA1 overexpression on luciferase activity of SOX4 reporter were evaluated in 786O cells. ( D ) The effect of miR129 downregulation and UCA1 silencing on luciferase activity of SOX4 reporter was monitored in ACHN cells. ( E ) SOX4 mRNA expression in 40 pairs of RCC tumor tissue and adjacent normal tissue. ( F ) Spearman’s correlation analysis of SOX4 mRNA and UCA1 expressions in 40 cases of RCC tumor tissue. * P

    Journal: OncoTargets and therapy

    Article Title: UCA1 promotes cell proliferation and invasion and inhibits apoptosis through regulation of the miR129–SOX4 pathway in renal cell carcinoma

    doi: 10.2147/OTT.S160192

    Figure Lengend Snippet: UCA1 acted as a ceRNA of miR129 to enhance target SOX4 gene expression in RCC cells. Notes: ( A ) 786O cells were transfected with miR129 mimic or miR-Con, and ACHN cells were transfected with miR129 inhibitor or anti-miR-Con, followed by the detection of SOX4 protein expression. ( B ) 786O cells were transfected with siCon or siUCA1-2, and ACHN cells were transfected with pcDNA3.1 vector or pcDNA-UCA1 plasmid, followed by the determination of SOX4 protein expression. ( C ) Effects of miR129 and UCA1 overexpression on luciferase activity of SOX4 reporter were evaluated in 786O cells. ( D ) The effect of miR129 downregulation and UCA1 silencing on luciferase activity of SOX4 reporter was monitored in ACHN cells. ( E ) SOX4 mRNA expression in 40 pairs of RCC tumor tissue and adjacent normal tissue. ( F ) Spearman’s correlation analysis of SOX4 mRNA and UCA1 expressions in 40 cases of RCC tumor tissue. * P

    Article Snippet: Full-length sequences of UCA1 were amplified by polymerase chain reaction (PCR) and inserted into pcDNA3.1 vector (Thermo Fisher Scientific) to gain the UCA1-overexpression plasmid.

    Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression, Luciferase, Activity Assay

    UCA1 inhibited miR129 expression in RCC cells by direct interaction. Notes: ( A ) Exhibition of putative binding sites between UCA1 and miR129 and mutant (Mut) sites in UCA1-Mut reporter. ( B ) 786O cells were cotransfected with miR control (Con) or miR129 mimic and wild-type (WT) UCA1 or UCA1-Mut reporter, followed by the detection of luciferase activity at 48 hours posttransfection. ( C ) Luciferase activity was measured in ACHN cells cotransfected with anti-miR-Con or miR129 inhibitor and UCA1-WT or UCA1-Mut reporter at 48 hours after transfection. ( D ) RNA pull-down assays were used to further validate the interaction between UCA1 and miR129 in 786O and ACHN cells. ( E , F ) RNA-immunoprecipitation assays were employed using Ago2 or IgG antibody to explore the enrichment degrees of UCA1 and miR129 in Ago2 or IgG immunoprecipitation complexes of 786O and ACHN cells. The input group acted as a positive control. ( G , H ) miR129 expression was determined in 786O and ACHN cells transfected with siCon ( G ), siUCA1-2 ( G ), UCA1 overexpression plasmid ( H ) or pcDNA3.1 empty vector ( H ). ( I ) miR129 expression analysis in 40 pairs of RCC tumor tissue and adjacent normal tissue. ( J ) Spearman’s correlation analysis of miR129 and UCA1 expression in 40 cases of RCC tumor tissue. * P

    Journal: OncoTargets and therapy

    Article Title: UCA1 promotes cell proliferation and invasion and inhibits apoptosis through regulation of the miR129–SOX4 pathway in renal cell carcinoma

    doi: 10.2147/OTT.S160192

    Figure Lengend Snippet: UCA1 inhibited miR129 expression in RCC cells by direct interaction. Notes: ( A ) Exhibition of putative binding sites between UCA1 and miR129 and mutant (Mut) sites in UCA1-Mut reporter. ( B ) 786O cells were cotransfected with miR control (Con) or miR129 mimic and wild-type (WT) UCA1 or UCA1-Mut reporter, followed by the detection of luciferase activity at 48 hours posttransfection. ( C ) Luciferase activity was measured in ACHN cells cotransfected with anti-miR-Con or miR129 inhibitor and UCA1-WT or UCA1-Mut reporter at 48 hours after transfection. ( D ) RNA pull-down assays were used to further validate the interaction between UCA1 and miR129 in 786O and ACHN cells. ( E , F ) RNA-immunoprecipitation assays were employed using Ago2 or IgG antibody to explore the enrichment degrees of UCA1 and miR129 in Ago2 or IgG immunoprecipitation complexes of 786O and ACHN cells. The input group acted as a positive control. ( G , H ) miR129 expression was determined in 786O and ACHN cells transfected with siCon ( G ), siUCA1-2 ( G ), UCA1 overexpression plasmid ( H ) or pcDNA3.1 empty vector ( H ). ( I ) miR129 expression analysis in 40 pairs of RCC tumor tissue and adjacent normal tissue. ( J ) Spearman’s correlation analysis of miR129 and UCA1 expression in 40 cases of RCC tumor tissue. * P

    Article Snippet: Full-length sequences of UCA1 were amplified by polymerase chain reaction (PCR) and inserted into pcDNA3.1 vector (Thermo Fisher Scientific) to gain the UCA1-overexpression plasmid.

    Techniques: Expressing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection, Immunoprecipitation, Positive Control, Over Expression, Plasmid Preparation

    SOCS1 physically interacts with RhTRIM5α. HEK293T cells were co-transfected with 1.0 µg of pRhTRIM5α-HA and 2.0 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 3.0 µg per sample with pcDNA3.1. Two days post-transfection, whole cell lysates were subjected to immunoprecipitation for RhTRIM5α with anti-HA antibody. Input lysates (left panels) and precipitated proteins (right panels) were separated by SDS-PAGE and proteins were detected by immunoblot analyses with anti-SOCS1 (upper panels) and anti-HA (RhTRIM5α-HA, lower panels) antibodies.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: SOCS1 physically interacts with RhTRIM5α. HEK293T cells were co-transfected with 1.0 µg of pRhTRIM5α-HA and 2.0 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 3.0 µg per sample with pcDNA3.1. Two days post-transfection, whole cell lysates were subjected to immunoprecipitation for RhTRIM5α with anti-HA antibody. Input lysates (left panels) and precipitated proteins (right panels) were separated by SDS-PAGE and proteins were detected by immunoblot analyses with anti-SOCS1 (upper panels) and anti-HA (RhTRIM5α-HA, lower panels) antibodies.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Transfection, Immunoprecipitation, SDS Page

    SOCS1 is detected in purified HIV-1 VLPs. HEK293T cells were co-transfected with 2.4 µg of pNL4-3, 2.4 µg of pRhTRIM5α-HA and 2.4 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1. (A) Relative viral titer in the supernatants was analyzed with TZM-bl indicator cells two days post-transfection. The titer of virus produced in the absence of RhTRIM5α and SOCS1 (Ct) was arbitrarily set as 100%. S1, T5α or T5α/S1 indicate virions produced in the presence of SOCS1 alone, RhTRIM5α-HA alone or both RhTRIM5α-HA and SOCS1, respectively. Producer cell lysates (B, left panels) and purified HIV-1 VLPs purified through a 20% sucrose layer (B, right panels) were subjected to immunoblot analysis. Proteins were detected with anti-HA (RhTRIM5α-HA, top panels), anti-SOCS1 (middle panels) and anti-HIV-1 p24 (bottom panels) antibodies.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: SOCS1 is detected in purified HIV-1 VLPs. HEK293T cells were co-transfected with 2.4 µg of pNL4-3, 2.4 µg of pRhTRIM5α-HA and 2.4 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1. (A) Relative viral titer in the supernatants was analyzed with TZM-bl indicator cells two days post-transfection. The titer of virus produced in the absence of RhTRIM5α and SOCS1 (Ct) was arbitrarily set as 100%. S1, T5α or T5α/S1 indicate virions produced in the presence of SOCS1 alone, RhTRIM5α-HA alone or both RhTRIM5α-HA and SOCS1, respectively. Producer cell lysates (B, left panels) and purified HIV-1 VLPs purified through a 20% sucrose layer (B, right panels) were subjected to immunoblot analysis. Proteins were detected with anti-HA (RhTRIM5α-HA, top panels), anti-SOCS1 (middle panels) and anti-HIV-1 p24 (bottom panels) antibodies.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Purification, Transfection, Produced

    SOCS1 affects RhTRIM5α expression in a dose-dependent manner. HEK293T cells were co-transfected with 0.1 µg of pNL4-3 or pUC18 with increasing amounts of pHuSOCS1 (0, 0.0375, 0.075, 0.15, 0.3 and 0.6 µg) and with or without 0.3 µg of pRhTRIM5α-HA. The amount of plasmid DNA was adjusted to 1.0 µg with pcDNA3.1. Two days post-transfection, viral titer in the culture supernatants was analyzed with TZM-bl indicator cells. (A) Relative viral titer obtained without exogenous SOCS1 and RhTRIM5α was arbitrarily set as 100%. The results are shown as an average of three independent experiments with standard deviation. (B) Whole cell lysates in panel (A) were subjected to immunoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (C) Relative band intensities of RhTRIM5α and SOCS1 in panel (B) were normalized with that of GAPDH. The normalized band intensity of RhTRIM5α (upper panels) in the absence of SOCS1 and that of SOCS1 (lower panels) without exogenous SOCS1 were arbitrarily set as 100%. (D) Effect of SOCS1 expression on transcriptional activity. HEK293T cell were co-transfected with 0.05 µg of pGL4.84-EF1α-hRlucCP (EF1α) or pcDNA3.1-Luc (CMV) together with 0.45 µg of pcDNA3.1 or pHuSOCS1. The amount of plasmid DNA was adjusted to 0.5 µg with pcDNA3.1. Two days after transfection, luciferase activity was determined and normalized with protein concentration (Luc/protein). The results are shown as an average of three independent experiments with standard deviation.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: SOCS1 affects RhTRIM5α expression in a dose-dependent manner. HEK293T cells were co-transfected with 0.1 µg of pNL4-3 or pUC18 with increasing amounts of pHuSOCS1 (0, 0.0375, 0.075, 0.15, 0.3 and 0.6 µg) and with or without 0.3 µg of pRhTRIM5α-HA. The amount of plasmid DNA was adjusted to 1.0 µg with pcDNA3.1. Two days post-transfection, viral titer in the culture supernatants was analyzed with TZM-bl indicator cells. (A) Relative viral titer obtained without exogenous SOCS1 and RhTRIM5α was arbitrarily set as 100%. The results are shown as an average of three independent experiments with standard deviation. (B) Whole cell lysates in panel (A) were subjected to immunoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (C) Relative band intensities of RhTRIM5α and SOCS1 in panel (B) were normalized with that of GAPDH. The normalized band intensity of RhTRIM5α (upper panels) in the absence of SOCS1 and that of SOCS1 (lower panels) without exogenous SOCS1 were arbitrarily set as 100%. (D) Effect of SOCS1 expression on transcriptional activity. HEK293T cell were co-transfected with 0.05 µg of pGL4.84-EF1α-hRlucCP (EF1α) or pcDNA3.1-Luc (CMV) together with 0.45 µg of pcDNA3.1 or pHuSOCS1. The amount of plasmid DNA was adjusted to 0.5 µg with pcDNA3.1. Two days after transfection, luciferase activity was determined and normalized with protein concentration (Luc/protein). The results are shown as an average of three independent experiments with standard deviation.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Expressing, Transfection, Plasmid Preparation, Standard Deviation, Activity Assay, Luciferase, Protein Concentration

    Reduction of RhTRIM5α by SOCS1 is proteasome-independent. HEK293T cells were transfected with 0.1 µg of pNL4-3, 0.3 µg of pRhTRIM5α-HA with or without 0.6 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Twenty-four hours after transfection, cells were treated with 30 µM of MG115 (lanes 3 and 4) or 30 µM of MG132 (lanes 5 and 6) for 16 hours. Whole cell lysates were subjected to immunoblot analyses with anti-HA (RhTRIM5α-HA, top panel), anti-SOCS1 (middle panel), anti-IκBα and anti-GAPDH (as loading control, bottom panel) antibodies.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: Reduction of RhTRIM5α by SOCS1 is proteasome-independent. HEK293T cells were transfected with 0.1 µg of pNL4-3, 0.3 µg of pRhTRIM5α-HA with or without 0.6 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Twenty-four hours after transfection, cells were treated with 30 µM of MG115 (lanes 3 and 4) or 30 µM of MG132 (lanes 5 and 6) for 16 hours. Whole cell lysates were subjected to immunoblot analyses with anti-HA (RhTRIM5α-HA, top panel), anti-SOCS1 (middle panel), anti-IκBα and anti-GAPDH (as loading control, bottom panel) antibodies.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Transfection

    SOCS1 reverses RhTRIM5α-mediated late restriction of HIV-1 replication. (A) HEK293T cells were transfected with 0.1 µg of pNL4-3 and with or without 0.6 µg of pHuSOCS1 (S1) and/or 0.3 µg of pRhTRIM5α-HA (T5α). Ct represents transfection with the control vectors. T5α/S1 indicates that S1 and T5α were co-transfected besides pNL4-3. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Two days post-transfection, relative viral titer in the supernatants was analyzed with TZM-bl indicator cells. Average of results from four independent experiments is shown with standard deviation. (B) The amount of p24 antigen in the supernatants was quantified with p24-specific ELISA. Data were obtained from the same experimental sets shown in panel A. (C) Twenty-four μg of whole cell lysates were subjected to immnoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (D) Relative RhTRIM5α protein expression level was determined by densitometry analysis in panel (C). The band intensity for T5α was arbitrarily set as 100%. (E) Relative Rh trim5α mRNA expression determined by quantitative RT-PCR. The value for T5α was arbitrarily set as 100%. The results are shown as an average obtained in four independent experiments with standard deviation.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: SOCS1 reverses RhTRIM5α-mediated late restriction of HIV-1 replication. (A) HEK293T cells were transfected with 0.1 µg of pNL4-3 and with or without 0.6 µg of pHuSOCS1 (S1) and/or 0.3 µg of pRhTRIM5α-HA (T5α). Ct represents transfection with the control vectors. T5α/S1 indicates that S1 and T5α were co-transfected besides pNL4-3. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Two days post-transfection, relative viral titer in the supernatants was analyzed with TZM-bl indicator cells. Average of results from four independent experiments is shown with standard deviation. (B) The amount of p24 antigen in the supernatants was quantified with p24-specific ELISA. Data were obtained from the same experimental sets shown in panel A. (C) Twenty-four μg of whole cell lysates were subjected to immnoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (D) Relative RhTRIM5α protein expression level was determined by densitometry analysis in panel (C). The band intensity for T5α was arbitrarily set as 100%. (E) Relative Rh trim5α mRNA expression determined by quantitative RT-PCR. The value for T5α was arbitrarily set as 100%. The results are shown as an average obtained in four independent experiments with standard deviation.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Transfection, Standard Deviation, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR