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pcdna3 1 neo vector  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pcdna3 1 neo vector
    Pcdna3 1 Neo Vector, supplied by Thermo Fisher, 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/product/pcdna3+1+neo+vector/bio_rxiv__2023__06__27__546668-195-8-10
    Average 86 stars, based on 1 article reviews
    pcdna3 1 neo vector - by Bioz Stars, 2026-09
    86/100 stars

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    Related Articles

    Plasmid Preparation:

    Article Title: Oligomeric Structure and Minimal Functional Unit of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1-A
    Article Snippet: .. The two individual units were linked through a linker peptide (Thr-Gly) in pcDNA3.1/neo vector (Invitrogen). .. To insert the linker peptide, the first unit (either wt-NBCe1-A or the T442C mutant) was cloned in pcDNA 3.1 vector (Invitrogen) between EcoRI and XhoI sites.

    Article Title: FGF-21/FGF-21 receptor interaction and activation is determined by betaKlotho.
    Article Snippet: Fibroblast growth factor-21 (FGF-21) is ametabolic regulator that can influence glucose and lipid control in diabetic rodents and primates.. We demonstrate that bKlotho is an integral part of an activated FGF-21-bKlotho-FGF receptor (FGFR) complex thus a critical subunit of the FGF-21 receptor.. Cells lacking bKlotho did not respond to FGF-21; the introduction of bKlotho to these cells conferred FGF-21-responsiveness and recapitulated the entire scope of FGF-21 signaling observed in naturally responsive cells.

    Article Title: Dimerization of the cellular prion protein inhibits propagation of scrapie prions
    Article Snippet: .. All constructs described above were inserted into pcDNA3.1/Neo (+) vector (Invitrogen). ..

    Article Title: The peroxisomal membrane protein import receptor Pex3p is directly transported to peroxisomes by a novel Pex19p- and Pex16p-dependent pathway
    Article Snippet: .. KpnI–XbaI fragment of the respective second PCR products was inserted into KpnI–XbaI sites of pcDNA3.1/Neo(+) vector (Invitrogen). ..

    Article Title: The pericentromeric protein shugoshin 2 cooperates with HSF 1 in heat shock response and RNA Pol II recruitment
    Article Snippet: .. DNA pull‐down assay A DNA fragment of human HSPA1A ( HSP70‐1 ) promoter (−448 to +13) was amplified by PCR using the pHSP70‐luc reporter plasmid as a template (Takii et al , 2010 ) and inserted into pcDNA3.1/Neo vector (Invitrogen) at the Kpn I/ Xho I site to generate phHSP70pro. ..

    Article Title: Nuclear quality control of non-imported secretory proteins attenuates proteostasis decline in the cytosol
    Article Snippet: .. All the mammalian expression plasmids were cloned into pcDNA3.1(+)-Neo vector (Invitrogen TM V79020). ..

    Sequencing:

    Article Title: FGF-21/FGF-21 receptor interaction and activation is determined by betaKlotho.
    Article Snippet: Fibroblast growth factor-21 (FGF-21) is ametabolic regulator that can influence glucose and lipid control in diabetic rodents and primates.. We demonstrate that bKlotho is an integral part of an activated FGF-21-bKlotho-FGF receptor (FGFR) complex thus a critical subunit of the FGF-21 receptor.. Cells lacking bKlotho did not respond to FGF-21; the introduction of bKlotho to these cells conferred FGF-21-responsiveness and recapitulated the entire scope of FGF-21 signaling observed in naturally responsive cells.

    Amplification:

    Article Title: FGF-21/FGF-21 receptor interaction and activation is determined by betaKlotho.
    Article Snippet: Fibroblast growth factor-21 (FGF-21) is ametabolic regulator that can influence glucose and lipid control in diabetic rodents and primates.. We demonstrate that bKlotho is an integral part of an activated FGF-21-bKlotho-FGF receptor (FGFR) complex thus a critical subunit of the FGF-21 receptor.. Cells lacking bKlotho did not respond to FGF-21; the introduction of bKlotho to these cells conferred FGF-21-responsiveness and recapitulated the entire scope of FGF-21 signaling observed in naturally responsive cells.

    Article Title: The pericentromeric protein shugoshin 2 cooperates with HSF 1 in heat shock response and RNA Pol II recruitment
    Article Snippet: .. DNA pull‐down assay A DNA fragment of human HSPA1A ( HSP70‐1 ) promoter (−448 to +13) was amplified by PCR using the pHSP70‐luc reporter plasmid as a template (Takii et al , 2010 ) and inserted into pcDNA3.1/Neo vector (Invitrogen) at the Kpn I/ Xho I site to generate phHSP70pro. ..

    Clone Assay:

    Article Title: FGF-21/FGF-21 receptor interaction and activation is determined by betaKlotho.
    Article Snippet: Fibroblast growth factor-21 (FGF-21) is ametabolic regulator that can influence glucose and lipid control in diabetic rodents and primates.. We demonstrate that bKlotho is an integral part of an activated FGF-21-bKlotho-FGF receptor (FGFR) complex thus a critical subunit of the FGF-21 receptor.. Cells lacking bKlotho did not respond to FGF-21; the introduction of bKlotho to these cells conferred FGF-21-responsiveness and recapitulated the entire scope of FGF-21 signaling observed in naturally responsive cells.

    Article Title: Nuclear quality control of non-imported secretory proteins attenuates proteostasis decline in the cytosol
    Article Snippet: .. All the mammalian expression plasmids were cloned into pcDNA3.1(+)-Neo vector (Invitrogen TM V79020). ..

    Expressing:

    Article Title: FGF-21/FGF-21 receptor interaction and activation is determined by betaKlotho.
    Article Snippet: Fibroblast growth factor-21 (FGF-21) is ametabolic regulator that can influence glucose and lipid control in diabetic rodents and primates.. We demonstrate that bKlotho is an integral part of an activated FGF-21-bKlotho-FGF receptor (FGFR) complex thus a critical subunit of the FGF-21 receptor.. Cells lacking bKlotho did not respond to FGF-21; the introduction of bKlotho to these cells conferred FGF-21-responsiveness and recapitulated the entire scope of FGF-21 signaling observed in naturally responsive cells.

    Article Title: Nuclear quality control of non-imported secretory proteins attenuates proteostasis decline in the cytosol
    Article Snippet: .. All the mammalian expression plasmids were cloned into pcDNA3.1(+)-Neo vector (Invitrogen TM V79020). ..

    Generated:

    Article Title: A Thr/Ser dual residue motif in the cytoplasmic tail of human CD1d is important for the down-regulation of antigen presentation following a herpes simplex virus 1 infection
    Article Snippet: .. 33 The CD1d mutants were generated by site-directed mutagenesis of the WT human CD1d (hCD1d) cDNA in the pcDNA3.1-neo vector (Invitrogen, Carlsbad, CA). ..

    Mutagenesis:

    Article Title: A Thr/Ser dual residue motif in the cytoplasmic tail of human CD1d is important for the down-regulation of antigen presentation following a herpes simplex virus 1 infection
    Article Snippet: .. 33 The CD1d mutants were generated by site-directed mutagenesis of the WT human CD1d (hCD1d) cDNA in the pcDNA3.1-neo vector (Invitrogen, Carlsbad, CA). ..

    Construct:

    Article Title: Dimerization of the cellular prion protein inhibits propagation of scrapie prions
    Article Snippet: .. All constructs described above were inserted into pcDNA3.1/Neo (+) vector (Invitrogen). ..

    Article Title: Biotin-ligase system for secretion of biotinylated protein
    Article Snippet: The gel purified PCR product was inserted downstream from the anti-CEA Db (Wu et al., 1999) in the pEE12 mammalian expression vector (Lonza Biologics, Slough, UK) (Bebbington et al., 1992) using SpeI and EcoRI sites. .. The resulting construct Db-BD123 was also inserted into the pcDNA3.1 neo vector (Invitrogen, Carlsbad, Calif.) using XbaI and EcoRI sites. ..

    Polymerase Chain Reaction:

    Article Title: The peroxisomal membrane protein import receptor Pex3p is directly transported to peroxisomes by a novel Pex19p- and Pex16p-dependent pathway
    Article Snippet: .. KpnI–XbaI fragment of the respective second PCR products was inserted into KpnI–XbaI sites of pcDNA3.1/Neo(+) vector (Invitrogen). ..

    Article Title: The pericentromeric protein shugoshin 2 cooperates with HSF 1 in heat shock response and RNA Pol II recruitment
    Article Snippet: .. DNA pull‐down assay A DNA fragment of human HSPA1A ( HSP70‐1 ) promoter (−448 to +13) was amplified by PCR using the pHSP70‐luc reporter plasmid as a template (Takii et al , 2010 ) and inserted into pcDNA3.1/Neo vector (Invitrogen) at the Kpn I/ Xho I site to generate phHSP70pro. ..

    Pull Down Assay:

    Article Title: The pericentromeric protein shugoshin 2 cooperates with HSF 1 in heat shock response and RNA Pol II recruitment
    Article Snippet: .. DNA pull‐down assay A DNA fragment of human HSPA1A ( HSP70‐1 ) promoter (−448 to +13) was amplified by PCR using the pHSP70‐luc reporter plasmid as a template (Takii et al , 2010 ) and inserted into pcDNA3.1/Neo vector (Invitrogen) at the Kpn I/ Xho I site to generate phHSP70pro. ..



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    A , HeLa cells were treated with a mock water control or 0.6 μM of the TBK1/IKKe inhibitor MRT67307 for 1.5 hours, then treated with TNFα (8 ng/mL) or PMA (10 ng/mL), or untreated (UN) and harvested after 5 minutes (TNFα) or 20 minutes (PMA and UN). Lysates (25 μg) were resolved by SDS-PAGE PAGE and probed for phospho-p65 (S536) and p65. The image shown is representative of at least two independent experiments. B-C , HeLa cells were treated with a mock water control or 0.6 μM of the TBK1/IKKe inhibitor MRT67307 for 1.5 hours, then transfected with 10 μM 2’3’-cGAM(PS) 2 (Rp/Sp) and Trans IT-LT1 at a 1:1 μg/μL ratio, or dsDNA (2 μg/mL) and Trans IT-LT1 at a 1:2 μg/μL ratio, or untreated (UN). Cells were harvested 5 hours after treatment, and lysates were resolved with SDS-PAGE and probed for p-p65 (S536) or p65 as in A (25 μg lysate loaded) or for <t>p-IRF3</t> (S386) and IRF3 (100 μg lysate loaded). Image shown is representative of at least two independent experiments.
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    Image Search Results


    A , HeLa cells were treated with a mock water control or 0.6 μM of the TBK1/IKKe inhibitor MRT67307 for 1.5 hours, then treated with TNFα (8 ng/mL) or PMA (10 ng/mL), or untreated (UN) and harvested after 5 minutes (TNFα) or 20 minutes (PMA and UN). Lysates (25 μg) were resolved by SDS-PAGE PAGE and probed for phospho-p65 (S536) and p65. The image shown is representative of at least two independent experiments. B-C , HeLa cells were treated with a mock water control or 0.6 μM of the TBK1/IKKe inhibitor MRT67307 for 1.5 hours, then transfected with 10 μM 2’3’-cGAM(PS) 2 (Rp/Sp) and Trans IT-LT1 at a 1:1 μg/μL ratio, or dsDNA (2 μg/mL) and Trans IT-LT1 at a 1:2 μg/μL ratio, or untreated (UN). Cells were harvested 5 hours after treatment, and lysates were resolved with SDS-PAGE and probed for p-p65 (S536) or p65 as in A (25 μg lysate loaded) or for p-IRF3 (S386) and IRF3 (100 μg lysate loaded). Image shown is representative of at least two independent experiments.

    Journal: bioRxiv

    Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

    doi: 10.1101/2022.03.08.483519

    Figure Lengend Snippet: A , HeLa cells were treated with a mock water control or 0.6 μM of the TBK1/IKKe inhibitor MRT67307 for 1.5 hours, then treated with TNFα (8 ng/mL) or PMA (10 ng/mL), or untreated (UN) and harvested after 5 minutes (TNFα) or 20 minutes (PMA and UN). Lysates (25 μg) were resolved by SDS-PAGE PAGE and probed for phospho-p65 (S536) and p65. The image shown is representative of at least two independent experiments. B-C , HeLa cells were treated with a mock water control or 0.6 μM of the TBK1/IKKe inhibitor MRT67307 for 1.5 hours, then transfected with 10 μM 2’3’-cGAM(PS) 2 (Rp/Sp) and Trans IT-LT1 at a 1:1 μg/μL ratio, or dsDNA (2 μg/mL) and Trans IT-LT1 at a 1:2 μg/μL ratio, or untreated (UN). Cells were harvested 5 hours after treatment, and lysates were resolved with SDS-PAGE and probed for p-p65 (S536) or p65 as in A (25 μg lysate loaded) or for p-IRF3 (S386) and IRF3 (100 μg lysate loaded). Image shown is representative of at least two independent experiments.

    Article Snippet: pcDNA3.1(+)-neo was obtained from ThermoFisher (Invitrogen cat. V790-20). pcDNA3.1(+)-neo-IRF3-5D was created using the wild-type template Human V5-IRF3-pcDNA3, a gift from Saumen Sarkar (Addgene plasmid # 32713; http://n2t.net/addgene:32713 ; RRID:Addgene_32713, ( )) and the Q5 site-directed mutagenesis kit (NEB, cat. E0554S) with the forward and reverse primers 5’-CTCGATCTCGACGATGACCAGTACAAGGCCTAC and 5’-TGGGTGGTCGTTGTCAATGTGCAGGTCCACAGT respectively; mutations were confirmed with sequencing. pCpGf-Bsr-GFP was constructed by PCR-amplifying GFP obtained from pSELECT-zeo-GFPBsr (Invivogen, cat. psetz-zgfpbsr) with a Kozak sequence encoded on the forward primer, then inserting it into pCpGfree-vitroBmcs (Invivogen, cat. pcpgvtb-mcsg2) digested with BglII and ApaL1; insertion was confirmed with sequencing. pcDNA3.1(+)puro was previously described ( ). pCpGfree-mcs was obtained from Invivogen (cat. pcpgf-mcs).

    Techniques: SDS Page, Transfection

    A , HeLa cells were seeded in 12-well plates to achieve ∼ 65% confluency in 24 hours, at which time cells were treated with either 8 ng/mL TNFα or a water vehicle control. Cells were harvested 24 hours after treatment, and RTqPCR was performed. Resulting levels of CCL22 mRNA are shown. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with an unpaired, two-tail t test; error bars represent standard deviations. B , HeLa cells were seeded and grown as for A and treated with either 10 ng/mL PMA or a 0.0004% DMSO vehicle control. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. C , HeLa cells expressing no shRNA (NS) or stably expressing a non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were harvested for RTqPCR; levels of RELA/p65 RNA are shown relative to untreated (NS) cells. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. D , Lysates (20 μg) from HeLa cells carrying no shRNA (NS), non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were resolved using SDS-PAGE and probed for RELA/p65 and beta-tubulin. The image shown is representative of at least three independent experiments. E , HeLa cells described in C and D were transfected with a mock control or dsDNA (2 μg/mL) with Trans IT-LT1 at a 1:2 ratio and harvested after 48 hours for RTqPCR. Resulting fold change of CCL22 mRNA is shown relative to the mock control for each individual cell line. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Dunnett’s pairwise comparison of each shRNA group to the control non-targeting group; note that this analysis was performed alongside the IRF3 shRNA groups from in order that all non-targeting control experiments be included; error bars represent standard deviations. F , HeLa cells carrying the shRNAs described above were transfected as in E in parallel experiments using a GFP expression plasmid (2 μg/mL, Trans IT-LT1 1:2 ratio) and imaged 48 hours after transfection. Brightfield (BF) shows the confluency of cells in the same field of view as GFP.

    Journal: bioRxiv

    Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

    doi: 10.1101/2022.03.08.483519

    Figure Lengend Snippet: A , HeLa cells were seeded in 12-well plates to achieve ∼ 65% confluency in 24 hours, at which time cells were treated with either 8 ng/mL TNFα or a water vehicle control. Cells were harvested 24 hours after treatment, and RTqPCR was performed. Resulting levels of CCL22 mRNA are shown. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with an unpaired, two-tail t test; error bars represent standard deviations. B , HeLa cells were seeded and grown as for A and treated with either 10 ng/mL PMA or a 0.0004% DMSO vehicle control. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. C , HeLa cells expressing no shRNA (NS) or stably expressing a non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were harvested for RTqPCR; levels of RELA/p65 RNA are shown relative to untreated (NS) cells. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. D , Lysates (20 μg) from HeLa cells carrying no shRNA (NS), non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were resolved using SDS-PAGE and probed for RELA/p65 and beta-tubulin. The image shown is representative of at least three independent experiments. E , HeLa cells described in C and D were transfected with a mock control or dsDNA (2 μg/mL) with Trans IT-LT1 at a 1:2 ratio and harvested after 48 hours for RTqPCR. Resulting fold change of CCL22 mRNA is shown relative to the mock control for each individual cell line. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Dunnett’s pairwise comparison of each shRNA group to the control non-targeting group; note that this analysis was performed alongside the IRF3 shRNA groups from in order that all non-targeting control experiments be included; error bars represent standard deviations. F , HeLa cells carrying the shRNAs described above were transfected as in E in parallel experiments using a GFP expression plasmid (2 μg/mL, Trans IT-LT1 1:2 ratio) and imaged 48 hours after transfection. Brightfield (BF) shows the confluency of cells in the same field of view as GFP.

    Article Snippet: pcDNA3.1(+)-neo was obtained from ThermoFisher (Invitrogen cat. V790-20). pcDNA3.1(+)-neo-IRF3-5D was created using the wild-type template Human V5-IRF3-pcDNA3, a gift from Saumen Sarkar (Addgene plasmid # 32713; http://n2t.net/addgene:32713 ; RRID:Addgene_32713, ( )) and the Q5 site-directed mutagenesis kit (NEB, cat. E0554S) with the forward and reverse primers 5’-CTCGATCTCGACGATGACCAGTACAAGGCCTAC and 5’-TGGGTGGTCGTTGTCAATGTGCAGGTCCACAGT respectively; mutations were confirmed with sequencing. pCpGf-Bsr-GFP was constructed by PCR-amplifying GFP obtained from pSELECT-zeo-GFPBsr (Invivogen, cat. psetz-zgfpbsr) with a Kozak sequence encoded on the forward primer, then inserting it into pCpGfree-vitroBmcs (Invivogen, cat. pcpgvtb-mcsg2) digested with BglII and ApaL1; insertion was confirmed with sequencing. pcDNA3.1(+)puro was previously described ( ). pCpGfree-mcs was obtained from Invivogen (cat. pcpgf-mcs).

    Techniques: Derivative Assay, Expressing, shRNA, Stable Transfection, SDS Page, Transfection, Plasmid Preparation

    A , HeLa cells expressing no shRNA (NS) or stably expressing a non-targeting shRNA (NT) or shRNAs against IRF3 (IRF3-1 or IRF3-2) were harvested for RTqPCR; levels of IRF3 RNA are shown relative to untreated (NS) cells. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. B , Lysates (20 μg) from HeLa cells carrying no shRNA (NS), non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were resolved using SDS-PAGE and probed for RELA/p65 and beta-tubulin. The image shown is representative of at least three independent experiments. C , HeLa cells described in A and B were transfected with a mock control or dsDNA (2 μg/mL) with Trans IT-LT1 at a 1:2 ratio and harvested after 48 hours for RTqPCR. Resulting fold change of CCL22 mRNA is shown relative to the mock control for each individual cell line. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Dunnett’s pairwise comparison of each shRNA group to the control non-targeting group; note that this analysis was performed alongside the RELA/p65 shRNA groups from in order that all non-targeting control experiments be included; error bars represent standard deviations. D , HeLa cells carrying the shRNAs described above were transfected as in C in parallel experiments using a GFP expression plasmid (2 μg/mL, Trans IT-LT1, 1:2 ratio) and imaged 48 hours after transfection. Brightfield (BF) shows the confluency of cells in the same field of view as GFP. E-F , HeLa cells ( E ) and MCF7 cells ( F ) were transfected with a mock control or 2 μg/mL of empty plasmid (EV) or the constitutively active IRF3-5D with Trans IT-LT1 (HeLa) at a 1:2 ratio or TransfeX (MCF7) at a 1:4 ratio. Cells were harvested 48 hours after transfection, and RTqPCR was performed. Resulting fold change of CCL22 mRNA relative to the mock control is shown. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with an unpaired, one-tailed t test; error bars represent standard deviations.

    Journal: bioRxiv

    Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

    doi: 10.1101/2022.03.08.483519

    Figure Lengend Snippet: A , HeLa cells expressing no shRNA (NS) or stably expressing a non-targeting shRNA (NT) or shRNAs against IRF3 (IRF3-1 or IRF3-2) were harvested for RTqPCR; levels of IRF3 RNA are shown relative to untreated (NS) cells. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. B , Lysates (20 μg) from HeLa cells carrying no shRNA (NS), non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were resolved using SDS-PAGE and probed for RELA/p65 and beta-tubulin. The image shown is representative of at least three independent experiments. C , HeLa cells described in A and B were transfected with a mock control or dsDNA (2 μg/mL) with Trans IT-LT1 at a 1:2 ratio and harvested after 48 hours for RTqPCR. Resulting fold change of CCL22 mRNA is shown relative to the mock control for each individual cell line. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Dunnett’s pairwise comparison of each shRNA group to the control non-targeting group; note that this analysis was performed alongside the RELA/p65 shRNA groups from in order that all non-targeting control experiments be included; error bars represent standard deviations. D , HeLa cells carrying the shRNAs described above were transfected as in C in parallel experiments using a GFP expression plasmid (2 μg/mL, Trans IT-LT1, 1:2 ratio) and imaged 48 hours after transfection. Brightfield (BF) shows the confluency of cells in the same field of view as GFP. E-F , HeLa cells ( E ) and MCF7 cells ( F ) were transfected with a mock control or 2 μg/mL of empty plasmid (EV) or the constitutively active IRF3-5D with Trans IT-LT1 (HeLa) at a 1:2 ratio or TransfeX (MCF7) at a 1:4 ratio. Cells were harvested 48 hours after transfection, and RTqPCR was performed. Resulting fold change of CCL22 mRNA relative to the mock control is shown. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with an unpaired, one-tailed t test; error bars represent standard deviations.

    Article Snippet: pcDNA3.1(+)-neo was obtained from ThermoFisher (Invitrogen cat. V790-20). pcDNA3.1(+)-neo-IRF3-5D was created using the wild-type template Human V5-IRF3-pcDNA3, a gift from Saumen Sarkar (Addgene plasmid # 32713; http://n2t.net/addgene:32713 ; RRID:Addgene_32713, ( )) and the Q5 site-directed mutagenesis kit (NEB, cat. E0554S) with the forward and reverse primers 5’-CTCGATCTCGACGATGACCAGTACAAGGCCTAC and 5’-TGGGTGGTCGTTGTCAATGTGCAGGTCCACAGT respectively; mutations were confirmed with sequencing. pCpGf-Bsr-GFP was constructed by PCR-amplifying GFP obtained from pSELECT-zeo-GFPBsr (Invivogen, cat. psetz-zgfpbsr) with a Kozak sequence encoded on the forward primer, then inserting it into pCpGfree-vitroBmcs (Invivogen, cat. pcpgvtb-mcsg2) digested with BglII and ApaL1; insertion was confirmed with sequencing. pcDNA3.1(+)puro was previously described ( ). pCpGfree-mcs was obtained from Invivogen (cat. pcpgf-mcs).

    Techniques: Expressing, shRNA, Stable Transfection, Derivative Assay, SDS Page, Transfection, Plasmid Preparation, One-tailed Test

    Cells were untreated (UN) or transfected with dsDNA (2 μg/mL) using Trans IT-LT1 (JEG-3, 1:3 ratio; HCT 116, 1:4 ratio) or TransfeX (MCF7, 1:4 ratio). Cells were harvested 48 hours after transfection. Lysates (100 μg) were separated with SDS-PAGE and probed for phospho-IRF3 (S386), IRF3, and beta-tubulin. The image shown is representative of at least two independent experiments.

    Journal: bioRxiv

    Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

    doi: 10.1101/2022.03.08.483519

    Figure Lengend Snippet: Cells were untreated (UN) or transfected with dsDNA (2 μg/mL) using Trans IT-LT1 (JEG-3, 1:3 ratio; HCT 116, 1:4 ratio) or TransfeX (MCF7, 1:4 ratio). Cells were harvested 48 hours after transfection. Lysates (100 μg) were separated with SDS-PAGE and probed for phospho-IRF3 (S386), IRF3, and beta-tubulin. The image shown is representative of at least two independent experiments.

    Article Snippet: pcDNA3.1(+)-neo was obtained from ThermoFisher (Invitrogen cat. V790-20). pcDNA3.1(+)-neo-IRF3-5D was created using the wild-type template Human V5-IRF3-pcDNA3, a gift from Saumen Sarkar (Addgene plasmid # 32713; http://n2t.net/addgene:32713 ; RRID:Addgene_32713, ( )) and the Q5 site-directed mutagenesis kit (NEB, cat. E0554S) with the forward and reverse primers 5’-CTCGATCTCGACGATGACCAGTACAAGGCCTAC and 5’-TGGGTGGTCGTTGTCAATGTGCAGGTCCACAGT respectively; mutations were confirmed with sequencing. pCpGf-Bsr-GFP was constructed by PCR-amplifying GFP obtained from pSELECT-zeo-GFPBsr (Invivogen, cat. psetz-zgfpbsr) with a Kozak sequence encoded on the forward primer, then inserting it into pCpGfree-vitroBmcs (Invivogen, cat. pcpgvtb-mcsg2) digested with BglII and ApaL1; insertion was confirmed with sequencing. pcDNA3.1(+)puro was previously described ( ). pCpGfree-mcs was obtained from Invivogen (cat. pcpgf-mcs).

    Techniques: Transfection, SDS Page