Structured Review

Thermo Fisher pcdna 3
ORF 57 increases ORF 59/58 mRNA and protein levels. (A) Top, autoradiogram of a Northern blot containing total RNA from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 59/58 in the presence (+) or absence (−) of 0.1 μg of pcDNA 3.1 ORF 57. A ds DNA probe specific for ORF 59 was used; bottom, the blot was reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific probe as a loading control. (B) Western blot analysis of protein extracts from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 59/58 in the presence (+) or absence (−) of 1 μg of pcDNA 3.1 ORF 57. The ORF 59 protein was detected with a KSHV MAb, and then with a secondary goat anti-mouse conjugated to horseradish peroxidase and ECL (Amersham). Equal amounts (10 μg) of extracts as determined by Bradford assay from each transfection were loaded onto the gel.
Pcdna 3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities †"

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities †

Journal: Journal of Virology

doi:

ORF 57 increases ORF 59/58 mRNA and protein levels. (A) Top, autoradiogram of a Northern blot containing total RNA from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 59/58 in the presence (+) or absence (−) of 0.1 μg of pcDNA 3.1 ORF 57. A ds DNA probe specific for ORF 59 was used; bottom, the blot was reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific probe as a loading control. (B) Western blot analysis of protein extracts from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 59/58 in the presence (+) or absence (−) of 1 μg of pcDNA 3.1 ORF 57. The ORF 59 protein was detected with a KSHV MAb, and then with a secondary goat anti-mouse conjugated to horseradish peroxidase and ECL (Amersham). Equal amounts (10 μg) of extracts as determined by Bradford assay from each transfection were loaded onto the gel.
Figure Legend Snippet: ORF 57 increases ORF 59/58 mRNA and protein levels. (A) Top, autoradiogram of a Northern blot containing total RNA from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 59/58 in the presence (+) or absence (−) of 0.1 μg of pcDNA 3.1 ORF 57. A ds DNA probe specific for ORF 59 was used; bottom, the blot was reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific probe as a loading control. (B) Western blot analysis of protein extracts from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 59/58 in the presence (+) or absence (−) of 1 μg of pcDNA 3.1 ORF 57. The ORF 59 protein was detected with a KSHV MAb, and then with a secondary goat anti-mouse conjugated to horseradish peroxidase and ECL (Amersham). Equal amounts (10 μg) of extracts as determined by Bradford assay from each transfection were loaded onto the gel.

Techniques Used: Northern Blot, Transfection, Western Blot, Bradford Assay

). Forty-eight hours posttransfection, luciferase activity was measured and values were plotted that represented fold activation relative to that of ORF 50 alone, which was set to 1. (C) Mutations in the nut-1 promoter abolish synergy between ORF 50 and ORF 57. CV-1 cells were transfected with 2 μg of pcDNA 3 ORF 50, increasing amounts of ORF 57, and either 1 μg of the wild-type nut-1-706 reporter or the nut-1-706 mutant reporter. Forty-eight hours posttransfection, luciferase activity was measured and values were plotted that represent fold activation relative to that of ORF 50 alone, which was set to 1.
Figure Legend Snippet: ). Forty-eight hours posttransfection, luciferase activity was measured and values were plotted that represented fold activation relative to that of ORF 50 alone, which was set to 1. (C) Mutations in the nut-1 promoter abolish synergy between ORF 50 and ORF 57. CV-1 cells were transfected with 2 μg of pcDNA 3 ORF 50, increasing amounts of ORF 57, and either 1 μg of the wild-type nut-1-706 reporter or the nut-1-706 mutant reporter. Forty-eight hours posttransfection, luciferase activity was measured and values were plotted that represent fold activation relative to that of ORF 50 alone, which was set to 1.

Techniques Used: Luciferase, Activity Assay, Activation Assay, Transfection, Mutagenesis

The C-terminally tagged version of ORF 57 is localized to the nucleus. The panels show results of immunofluorescence staining of CV-1 cells 48 h after transfection with either empty pcDNA 3.1-V5 vector (left panel) or pcDNA 3.1-V5 ORF 57 (right panel). Reactivity to anti-V5 antibody (Invitrogen) was detected with a TRITC-conjugated rabbit anti-mouse antibody.
Figure Legend Snippet: The C-terminally tagged version of ORF 57 is localized to the nucleus. The panels show results of immunofluorescence staining of CV-1 cells 48 h after transfection with either empty pcDNA 3.1-V5 vector (left panel) or pcDNA 3.1-V5 ORF 57 (right panel). Reactivity to anti-V5 antibody (Invitrogen) was detected with a TRITC-conjugated rabbit anti-mouse antibody.

Techniques Used: Immunofluorescence, Staining, Transfection, Plasmid Preparation

ORF 50 mRNA and protein levels are not significantly increased by ORF 57 expression. (A) Top, autoradiogram of a Northern blot containing total RNA from CV-1 cells transfected with a fixed amount of pcDNA 3 ORF 50 and the following amounts of pcDNA 3.1 ORF 57: 0 μg (lane 1), 0.75 μg (lane 2), 2.5 μg (lane 3), and 7.5 μg (lane 4). An antisense riboprobe for ORF 50 was hybridized to the blot. Bottom, the blot was reprobed with a GAPDH-specific probe as a loading control. (B) Western blot analysis of protein extracts from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 50 and the following amounts of pcDNA 3.1 ORF 57: 0 μg (lane 1), 0.5 μg (lane 2), 2.5 μg (lane 3), and 5 μg (lane 4). The ORF 50 protein was detected with a polyclonal antibody and then with a secondary rabbit anti-mouse antibody conjugated to horseradish peroxidase and ECL (Amersham). Equal amount of extracts as determined by Bradford assay from each transfection were loaded onto the gel.
Figure Legend Snippet: ORF 50 mRNA and protein levels are not significantly increased by ORF 57 expression. (A) Top, autoradiogram of a Northern blot containing total RNA from CV-1 cells transfected with a fixed amount of pcDNA 3 ORF 50 and the following amounts of pcDNA 3.1 ORF 57: 0 μg (lane 1), 0.75 μg (lane 2), 2.5 μg (lane 3), and 7.5 μg (lane 4). An antisense riboprobe for ORF 50 was hybridized to the blot. Bottom, the blot was reprobed with a GAPDH-specific probe as a loading control. (B) Western blot analysis of protein extracts from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 50 and the following amounts of pcDNA 3.1 ORF 57: 0 μg (lane 1), 0.5 μg (lane 2), 2.5 μg (lane 3), and 5 μg (lane 4). The ORF 50 protein was detected with a polyclonal antibody and then with a secondary rabbit anti-mouse antibody conjugated to horseradish peroxidase and ECL (Amersham). Equal amount of extracts as determined by Bradford assay from each transfection were loaded onto the gel.

Techniques Used: Expressing, Northern Blot, Transfection, Western Blot, Bradford Assay

Effect of introns on synergistic activation of different promoters by the ORF 50-ORF 57 combination. CV-1 cells were cotransfected with fixed amounts of the indicated KSHV promoter constructs with or without introns (Nut-1, TK, and Kaposin), a fixed amount of the transcriptional activator, pcDNA 3 ORF 50, and increasing amounts of pcDNA 3.1 ORF 57 (0, 0.3, 1, and 3 μg). Luciferase activity was measured 48 h after transfection. (A) Log scale graph displaying the effects on the Nut-1 promoter with and without an intron. (B) Graph displaying the effects on the Kaposin and TK promoters with and without an intron. Values are plotted as fold luciferase activity over that of ORF 50 alone, which was set to 1. Error bars representing standard deviations are from two experiments performed in duplicate. Note that activation is plotted on an arithmetic scale in B and on a log scale in A.
Figure Legend Snippet: Effect of introns on synergistic activation of different promoters by the ORF 50-ORF 57 combination. CV-1 cells were cotransfected with fixed amounts of the indicated KSHV promoter constructs with or without introns (Nut-1, TK, and Kaposin), a fixed amount of the transcriptional activator, pcDNA 3 ORF 50, and increasing amounts of pcDNA 3.1 ORF 57 (0, 0.3, 1, and 3 μg). Luciferase activity was measured 48 h after transfection. (A) Log scale graph displaying the effects on the Nut-1 promoter with and without an intron. (B) Graph displaying the effects on the Kaposin and TK promoters with and without an intron. Values are plotted as fold luciferase activity over that of ORF 50 alone, which was set to 1. Error bars representing standard deviations are from two experiments performed in duplicate. Note that activation is plotted on an arithmetic scale in B and on a log scale in A.

Techniques Used: Activation Assay, Construct, Luciferase, Activity Assay, Transfection

Effect of ORF 57 expression on viral promoter constructs with and without introns. CV-1 cells were cotransfected with increasing amounts of pcDNA 3.1 ORF 57 (0, 0.3, 1, and 3 μg) and fixed amounts of the following KSHV promoter luciferase constructs with or without an intron: DNA Pol, Kaposin, Nut-1, and TK. Luciferase activity was measured 48 h posttransfection. Graphs are representative examples of experiments performed in duplicate. Error bars represent standard deviations.
Figure Legend Snippet: Effect of ORF 57 expression on viral promoter constructs with and without introns. CV-1 cells were cotransfected with increasing amounts of pcDNA 3.1 ORF 57 (0, 0.3, 1, and 3 μg) and fixed amounts of the following KSHV promoter luciferase constructs with or without an intron: DNA Pol, Kaposin, Nut-1, and TK. Luciferase activity was measured 48 h posttransfection. Graphs are representative examples of experiments performed in duplicate. Error bars represent standard deviations.

Techniques Used: Expressing, Construct, Luciferase, Activity Assay

Upregulation of nut-1 RNA accumulation by ORF 57 expression. The indicated constructs (bottom) were transfected into CV-1 cells in the presence (+) or absence (−) of 1 μg of pcDNA 3.1 ORF 57. Forty-eight hours later, total RNA was prepared from whole-cell lysates, electrophoresed through 1% agarose-formaldehyde gels, transferred to Hybond-N+ membranes, and hybridized to probes specific for nut-1 (A) or ORF 74/GCR (B, left panel) or ORF K5 (B, right panel). Below each panel is shown rRNA bands in ethidium bromide-stained lanes as a loading control.
Figure Legend Snippet: Upregulation of nut-1 RNA accumulation by ORF 57 expression. The indicated constructs (bottom) were transfected into CV-1 cells in the presence (+) or absence (−) of 1 μg of pcDNA 3.1 ORF 57. Forty-eight hours later, total RNA was prepared from whole-cell lysates, electrophoresed through 1% agarose-formaldehyde gels, transferred to Hybond-N+ membranes, and hybridized to probes specific for nut-1 (A) or ORF 74/GCR (B, left panel) or ORF K5 (B, right panel). Below each panel is shown rRNA bands in ethidium bromide-stained lanes as a loading control.

Techniques Used: Expressing, Construct, Transfection, Staining

Related Articles

Clone Assay:

Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition
Article Snippet: .. Generation of cell lines stably expressing ACVR1-IPF The InversePericam FKBP1A (IPF) fusion protein was amplified by PCR from the pCS2+zALK3 IPF ( ) and cloned in-frame downstream of the human ACVR1 cDNA sequence in the pcDNA3.1 Hygro (+) vector (Thermo Fisher). .. MDCKII and NIH-3T3 cells were transfected with the ACVR1-IPF construct and selected with 400 µg/ml hygromycin or 40 µg/ml hygromycin, respectively.

Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells
Article Snippet: .. PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning. .. Digested products were separated on an agarose gel, extracted and purified (Qiagen).

Article Title: Nesprin-2 accumulates at the front of the nucleus during confined cell migration
Article Snippet: .. Creation of stable NIH 3T3 mini nesprin cell lines Plasmids were cloned into a pcDNA3.1 hygro(−) vector (ThermoFisher), digested by BamHI using a In-fusion HD cloning kit. .. The cytoplasmic and KASH domains of the mini-nesprin constructs were obtained from a previous publication.

Transfection:

Article Title: Cyclin D1 inhibits whereas c-Myc enhances the cytotoxicity of cisplatin in mouse pancreatic cancer cells via regulation of several members of the NF-?B and Bcl-2 families
Article Snippet: .. Ela-mycT1, MDA-MB-231 (MB231) and D1.5 cells were transiently transfected with pcDNA3.1-cyclin D1 (D1), pcDNA3.1-neo vector, pcDNA3.1-c-myc , pcDNA3.1-hygro vector, pSiD1shRNA and pSishRNA plasmids using LipofectAMINE reagent ( www.Invitrogen.com ) according to the manufacturer's protocol. .. In cases when cells were treated with CDDP after transfection, the medium was replaced with a fresh one containing the indicated concentration of CDDP every 24 hours.

Amplification:

Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition
Article Snippet: .. Generation of cell lines stably expressing ACVR1-IPF The InversePericam FKBP1A (IPF) fusion protein was amplified by PCR from the pCS2+zALK3 IPF ( ) and cloned in-frame downstream of the human ACVR1 cDNA sequence in the pcDNA3.1 Hygro (+) vector (Thermo Fisher). .. MDCKII and NIH-3T3 cells were transfected with the ACVR1-IPF construct and selected with 400 µg/ml hygromycin or 40 µg/ml hygromycin, respectively.

Article Title: Membrane Orientation and Subcellular Localization of Transmembrane Protein 106B (TMEM106B), a Major Risk Factor for Frontotemporal Lobar Degeneration *
Article Snippet: .. TMEM106B wild type (WT) cDNA was amplified by PCR and subcloned into the BamHI and XhoI restriction sites of the pcDNA 3.1/Hygro(+) or the pcDNATM4/TO expression vector (Invitrogen). .. The HA tag was introduced by a 5′- or 3′-primer.

Stable Transfection:

Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition
Article Snippet: .. Generation of cell lines stably expressing ACVR1-IPF The InversePericam FKBP1A (IPF) fusion protein was amplified by PCR from the pCS2+zALK3 IPF ( ) and cloned in-frame downstream of the human ACVR1 cDNA sequence in the pcDNA3.1 Hygro (+) vector (Thermo Fisher). .. MDCKII and NIH-3T3 cells were transfected with the ACVR1-IPF construct and selected with 400 µg/ml hygromycin or 40 µg/ml hygromycin, respectively.

Polymerase Chain Reaction:

Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition
Article Snippet: .. Generation of cell lines stably expressing ACVR1-IPF The InversePericam FKBP1A (IPF) fusion protein was amplified by PCR from the pCS2+zALK3 IPF ( ) and cloned in-frame downstream of the human ACVR1 cDNA sequence in the pcDNA3.1 Hygro (+) vector (Thermo Fisher). .. MDCKII and NIH-3T3 cells were transfected with the ACVR1-IPF construct and selected with 400 µg/ml hygromycin or 40 µg/ml hygromycin, respectively.

Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells
Article Snippet: .. PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning. .. Digested products were separated on an agarose gel, extracted and purified (Qiagen).

Article Title: Membrane Orientation and Subcellular Localization of Transmembrane Protein 106B (TMEM106B), a Major Risk Factor for Frontotemporal Lobar Degeneration *
Article Snippet: .. TMEM106B wild type (WT) cDNA was amplified by PCR and subcloned into the BamHI and XhoI restriction sites of the pcDNA 3.1/Hygro(+) or the pcDNATM4/TO expression vector (Invitrogen). .. The HA tag was introduced by a 5′- or 3′-primer.

Multiple Displacement Amplification:

Article Title: Cyclin D1 inhibits whereas c-Myc enhances the cytotoxicity of cisplatin in mouse pancreatic cancer cells via regulation of several members of the NF-?B and Bcl-2 families
Article Snippet: .. Ela-mycT1, MDA-MB-231 (MB231) and D1.5 cells were transiently transfected with pcDNA3.1-cyclin D1 (D1), pcDNA3.1-neo vector, pcDNA3.1-c-myc , pcDNA3.1-hygro vector, pSiD1shRNA and pSishRNA plasmids using LipofectAMINE reagent ( www.Invitrogen.com ) according to the manufacturer's protocol. .. In cases when cells were treated with CDDP after transfection, the medium was replaced with a fresh one containing the indicated concentration of CDDP every 24 hours.

Patch Clamp:

Article Title: Mechanism of Increased BK Channel Activation from a Channel Mutation that Causes Epilepsy
Article Snippet: .. Patch Clamp Recording of HEK Cells Experiments were performed with the mouse α subunit cDNA expression vector in pcDNA3 (GenBank accession no. MMU09383 ) and mouse β4 in the vector pcDNA3.1Hygro(+) (Invitrogen). .. The D369G mutation, originally described in the human gene (D434G in human) , was introduced in the mouse α subunit cDNA using the Quick-Change Mutagenesis kit (Agilent Technologies) and verified by sequencing.

FLAG-tag:

Article Title: Epithelial membrane protein 1 promotes tumor metastasis by enhancing cell migration via copine-III and Rac1
Article Snippet: .. EMP1 cDNA was subcloned into the pFLAG-CMV5 vector (Sigma-Aldrich), and then EMP1 cDNA including the FLAG-tag sequence was cut out and inserted in the pcDNA3.1-Hygro vector (Thermo Fisher Scientific, Waltham, MA, USA) to create the pcDNA3.1-Hygro-EMP1-FLAG vector. .. EGFP-tagged copine-III-WT (amino acids 1–537), copine-III-ΔC2-1 (amino acids 139–515), copine-III-ΔC2-2 (amino acids 7–125 and 263–515), and copine-III-ΔVWA (amino acids 7–251) were constructed with the pEGFP-C1 vector (BD Biosciences, San Jose, CA, USA).

Article Title: Mutant huntingtin Impairs the Post-Golgi Trafficking of Brain-Derived Neurotrophic Factor But Not Its Val66Met Polymorphism
Article Snippet: .. The human BDNF and its Val66Met polymorphism subcloned into pCDNA3.1hygro expression vector (Invitrogen) with hemagglutinin epitope tag at 3′ and FLAG epitope tag at 5′ was kindly supplied by Dr. Francis S. Lee (Weill Medical College of Cornell University, New York, NY) ( ). .. BDNF and its variant Val66Met polymorphism subcloned into enhanced green fluorescent protein (eGFP)–N1 plasmid (Clontech, Palo Alto, CA) was described by .

Expressing:

Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition
Article Snippet: .. Generation of cell lines stably expressing ACVR1-IPF The InversePericam FKBP1A (IPF) fusion protein was amplified by PCR from the pCS2+zALK3 IPF ( ) and cloned in-frame downstream of the human ACVR1 cDNA sequence in the pcDNA3.1 Hygro (+) vector (Thermo Fisher). .. MDCKII and NIH-3T3 cells were transfected with the ACVR1-IPF construct and selected with 400 µg/ml hygromycin or 40 µg/ml hygromycin, respectively.

Article Title: Mechanism of Increased BK Channel Activation from a Channel Mutation that Causes Epilepsy
Article Snippet: .. Patch Clamp Recording of HEK Cells Experiments were performed with the mouse α subunit cDNA expression vector in pcDNA3 (GenBank accession no. MMU09383 ) and mouse β4 in the vector pcDNA3.1Hygro(+) (Invitrogen). .. The D369G mutation, originally described in the human gene (D434G in human) , was introduced in the mouse α subunit cDNA using the Quick-Change Mutagenesis kit (Agilent Technologies) and verified by sequencing.

Article Title: Mutant huntingtin Impairs the Post-Golgi Trafficking of Brain-Derived Neurotrophic Factor But Not Its Val66Met Polymorphism
Article Snippet: .. The human BDNF and its Val66Met polymorphism subcloned into pCDNA3.1hygro expression vector (Invitrogen) with hemagglutinin epitope tag at 3′ and FLAG epitope tag at 5′ was kindly supplied by Dr. Francis S. Lee (Weill Medical College of Cornell University, New York, NY) ( ). .. BDNF and its variant Val66Met polymorphism subcloned into enhanced green fluorescent protein (eGFP)–N1 plasmid (Clontech, Palo Alto, CA) was described by .

Article Title: Membrane Orientation and Subcellular Localization of Transmembrane Protein 106B (TMEM106B), a Major Risk Factor for Frontotemporal Lobar Degeneration *
Article Snippet: .. TMEM106B wild type (WT) cDNA was amplified by PCR and subcloned into the BamHI and XhoI restriction sites of the pcDNA 3.1/Hygro(+) or the pcDNATM4/TO expression vector (Invitrogen). .. The HA tag was introduced by a 5′- or 3′-primer.

Sequencing:

Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition
Article Snippet: .. Generation of cell lines stably expressing ACVR1-IPF The InversePericam FKBP1A (IPF) fusion protein was amplified by PCR from the pCS2+zALK3 IPF ( ) and cloned in-frame downstream of the human ACVR1 cDNA sequence in the pcDNA3.1 Hygro (+) vector (Thermo Fisher). .. MDCKII and NIH-3T3 cells were transfected with the ACVR1-IPF construct and selected with 400 µg/ml hygromycin or 40 µg/ml hygromycin, respectively.

Article Title: Epithelial membrane protein 1 promotes tumor metastasis by enhancing cell migration via copine-III and Rac1
Article Snippet: .. EMP1 cDNA was subcloned into the pFLAG-CMV5 vector (Sigma-Aldrich), and then EMP1 cDNA including the FLAG-tag sequence was cut out and inserted in the pcDNA3.1-Hygro vector (Thermo Fisher Scientific, Waltham, MA, USA) to create the pcDNA3.1-Hygro-EMP1-FLAG vector. .. EGFP-tagged copine-III-WT (amino acids 1–537), copine-III-ΔC2-1 (amino acids 139–515), copine-III-ΔC2-2 (amino acids 7–125 and 263–515), and copine-III-ΔVWA (amino acids 7–251) were constructed with the pEGFP-C1 vector (BD Biosciences, San Jose, CA, USA).

Plasmid Preparation:

Article Title: TGF-β uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition
Article Snippet: .. Generation of cell lines stably expressing ACVR1-IPF The InversePericam FKBP1A (IPF) fusion protein was amplified by PCR from the pCS2+zALK3 IPF ( ) and cloned in-frame downstream of the human ACVR1 cDNA sequence in the pcDNA3.1 Hygro (+) vector (Thermo Fisher). .. MDCKII and NIH-3T3 cells were transfected with the ACVR1-IPF construct and selected with 400 µg/ml hygromycin or 40 µg/ml hygromycin, respectively.

Article Title: Epithelial membrane protein 1 promotes tumor metastasis by enhancing cell migration via copine-III and Rac1
Article Snippet: .. EMP1 cDNA was subcloned into the pFLAG-CMV5 vector (Sigma-Aldrich), and then EMP1 cDNA including the FLAG-tag sequence was cut out and inserted in the pcDNA3.1-Hygro vector (Thermo Fisher Scientific, Waltham, MA, USA) to create the pcDNA3.1-Hygro-EMP1-FLAG vector. .. EGFP-tagged copine-III-WT (amino acids 1–537), copine-III-ΔC2-1 (amino acids 139–515), copine-III-ΔC2-2 (amino acids 7–125 and 263–515), and copine-III-ΔVWA (amino acids 7–251) were constructed with the pEGFP-C1 vector (BD Biosciences, San Jose, CA, USA).

Article Title: Cyclin D1 inhibits whereas c-Myc enhances the cytotoxicity of cisplatin in mouse pancreatic cancer cells via regulation of several members of the NF-?B and Bcl-2 families
Article Snippet: .. Ela-mycT1, MDA-MB-231 (MB231) and D1.5 cells were transiently transfected with pcDNA3.1-cyclin D1 (D1), pcDNA3.1-neo vector, pcDNA3.1-c-myc , pcDNA3.1-hygro vector, pSiD1shRNA and pSishRNA plasmids using LipofectAMINE reagent ( www.Invitrogen.com ) according to the manufacturer's protocol. .. In cases when cells were treated with CDDP after transfection, the medium was replaced with a fresh one containing the indicated concentration of CDDP every 24 hours.

Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells
Article Snippet: .. PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning. .. Digested products were separated on an agarose gel, extracted and purified (Qiagen).

Article Title: Nesprin-2 accumulates at the front of the nucleus during confined cell migration
Article Snippet: .. Creation of stable NIH 3T3 mini nesprin cell lines Plasmids were cloned into a pcDNA3.1 hygro(−) vector (ThermoFisher), digested by BamHI using a In-fusion HD cloning kit. .. The cytoplasmic and KASH domains of the mini-nesprin constructs were obtained from a previous publication.

Article Title: Mechanism of Increased BK Channel Activation from a Channel Mutation that Causes Epilepsy
Article Snippet: .. Patch Clamp Recording of HEK Cells Experiments were performed with the mouse α subunit cDNA expression vector in pcDNA3 (GenBank accession no. MMU09383 ) and mouse β4 in the vector pcDNA3.1Hygro(+) (Invitrogen). .. The D369G mutation, originally described in the human gene (D434G in human) , was introduced in the mouse α subunit cDNA using the Quick-Change Mutagenesis kit (Agilent Technologies) and verified by sequencing.

Article Title: Mutant huntingtin Impairs the Post-Golgi Trafficking of Brain-Derived Neurotrophic Factor But Not Its Val66Met Polymorphism
Article Snippet: .. The human BDNF and its Val66Met polymorphism subcloned into pCDNA3.1hygro expression vector (Invitrogen) with hemagglutinin epitope tag at 3′ and FLAG epitope tag at 5′ was kindly supplied by Dr. Francis S. Lee (Weill Medical College of Cornell University, New York, NY) ( ). .. BDNF and its variant Val66Met polymorphism subcloned into enhanced green fluorescent protein (eGFP)–N1 plasmid (Clontech, Palo Alto, CA) was described by .

Article Title: Membrane Orientation and Subcellular Localization of Transmembrane Protein 106B (TMEM106B), a Major Risk Factor for Frontotemporal Lobar Degeneration *
Article Snippet: .. TMEM106B wild type (WT) cDNA was amplified by PCR and subcloned into the BamHI and XhoI restriction sites of the pcDNA 3.1/Hygro(+) or the pcDNATM4/TO expression vector (Invitrogen). .. The HA tag was introduced by a 5′- or 3′-primer.

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    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 1996 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna3 1 nkx2 1 as1 vector
    <t>NKX2-1-AS1</t> knockdown alters gene expression patterns in H441 cells. ( A ) List of the top 20 genes down- and (B) up-regulated by NKX2-1-AS1 knockdown determined by microarray analysis, 48 h after treatment (n = 6). ( C ) qPCR validation of down-regulated and up-regulated genes in NKX2-1-AS1 knockdown cells at 48 h after treatment (n = 3, *p
    Pcdna3 1 Nkx2 1 As1 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    PR130 expression antagonises ATM phosphorylation. a Schematic of PPP2R3A knockout using CRISPR-Cas9 technology. Humanised S. pyogenes Cas9 nuclease was expressed in HCT116 cells with two guide RNAs (gRNA) to introduce two DSB into the PPP2R3A gene (684 bp apart) as described in the Methods section. The usage of two cleavage sites increased the chances to create a successful knockout. b HCT116 ΔgRNA and HCT116 ΔPR130 (clone #16) cells were cultured with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. ATM, pATM (S1981), p53, pp53 (S15) and PR130 were detected by western blot. β-Actin served as loading control. Numbers indicate densitometric analysis of pATM signal relative to HU-treated sample of each cell line and normalised to β-Actin signal ( n = 3). c HCT116 cells were transiently transfected with indicated siRNAs. After 24 h, cells were treated with 1 mM HU and 2 µM MS-275 (24 h). Protein detection was performed by immunoblot ( n = 2). d After 20 h of treatment with HU (1 mM) and MS-275 (2 µM), okadaic acid (OA, 25 nM) was added for further 4 h. Co-immunoprecipitations (IP) with anti-pS1981-ATM antibody, rabbit pre-immune serum (pre) or no antibody (no AB) were analysed for pATM and PR130 by western blot. Input (IN) represents 6% of the lysates used for IP. The right panel shows results that were obtained with the same strategy, including individual treatment with HU and MS-275. Data represent results from three independent experiments that were carried out in a similar manner. e IP performed with PR130 antibody using lysates from untreated and MS-275-treated HCT116. Precipitates were probed with pan-acetyl-lysine (ac-Lys) and PR130 antibody. Input (IN) is 6% of the lysate used for immunoprecipitation ( n = 2). Numbers indicate densitometric analysis of ac-Lys and PR130 signal relative to untreated cells. f HCT116 cells were transfected with HA-PR130 or vector control (pcDNA3.1) for 24 h and subsequently treated with MS-275 (2 µM, 24 h). Phosphatase activity of the HA-precipitates against phospho-S1981-ATM peptide or a threonine phosphopeptide (positive control) were measured in vitro as liberated pmol phosphate/min. Data are presented as mean ± SEM ( n = 3). The western blot verifies precipitation of HA-tagged PR130

    Journal: Nature Communications

    Article Title: HDAC1 and HDAC2 integrate checkpoint kinase phosphorylation and cell fate through the phosphatase-2A subunit PR130

    doi: 10.1038/s41467-018-03096-0

    Figure Lengend Snippet: PR130 expression antagonises ATM phosphorylation. a Schematic of PPP2R3A knockout using CRISPR-Cas9 technology. Humanised S. pyogenes Cas9 nuclease was expressed in HCT116 cells with two guide RNAs (gRNA) to introduce two DSB into the PPP2R3A gene (684 bp apart) as described in the Methods section. The usage of two cleavage sites increased the chances to create a successful knockout. b HCT116 ΔgRNA and HCT116 ΔPR130 (clone #16) cells were cultured with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. ATM, pATM (S1981), p53, pp53 (S15) and PR130 were detected by western blot. β-Actin served as loading control. Numbers indicate densitometric analysis of pATM signal relative to HU-treated sample of each cell line and normalised to β-Actin signal ( n = 3). c HCT116 cells were transiently transfected with indicated siRNAs. After 24 h, cells were treated with 1 mM HU and 2 µM MS-275 (24 h). Protein detection was performed by immunoblot ( n = 2). d After 20 h of treatment with HU (1 mM) and MS-275 (2 µM), okadaic acid (OA, 25 nM) was added for further 4 h. Co-immunoprecipitations (IP) with anti-pS1981-ATM antibody, rabbit pre-immune serum (pre) or no antibody (no AB) were analysed for pATM and PR130 by western blot. Input (IN) represents 6% of the lysates used for IP. The right panel shows results that were obtained with the same strategy, including individual treatment with HU and MS-275. Data represent results from three independent experiments that were carried out in a similar manner. e IP performed with PR130 antibody using lysates from untreated and MS-275-treated HCT116. Precipitates were probed with pan-acetyl-lysine (ac-Lys) and PR130 antibody. Input (IN) is 6% of the lysate used for immunoprecipitation ( n = 2). Numbers indicate densitometric analysis of ac-Lys and PR130 signal relative to untreated cells. f HCT116 cells were transfected with HA-PR130 or vector control (pcDNA3.1) for 24 h and subsequently treated with MS-275 (2 µM, 24 h). Phosphatase activity of the HA-precipitates against phospho-S1981-ATM peptide or a threonine phosphopeptide (positive control) were measured in vitro as liberated pmol phosphate/min. Data are presented as mean ± SEM ( n = 3). The western blot verifies precipitation of HA-tagged PR130

    Article Snippet: HCT116 cells were transfected with HA-tagged PR130 or pcDNA3.1 (vector control) using TurboFect (ThermoFisher Scientific).

    Techniques: Expressing, Knock-Out, CRISPR, Introduce, Cell Culture, Mass Spectrometry, Western Blot, Transfection, Immunoprecipitation, Plasmid Preparation, Activity Assay, Positive Control, In Vitro

    NKX2-1-AS1 knockdown alters gene expression patterns in H441 cells. ( A ) List of the top 20 genes down- and (B) up-regulated by NKX2-1-AS1 knockdown determined by microarray analysis, 48 h after treatment (n = 6). ( C ) qPCR validation of down-regulated and up-regulated genes in NKX2-1-AS1 knockdown cells at 48 h after treatment (n = 3, *p

    Journal: Scientific Reports

    Article Title: NKX2-1-AS1 negatively regulates CD274/PD-L1, cell-cell interaction genes, and limits human lung carcinoma cell migration

    doi: 10.1038/s41598-018-32793-5

    Figure Lengend Snippet: NKX2-1-AS1 knockdown alters gene expression patterns in H441 cells. ( A ) List of the top 20 genes down- and (B) up-regulated by NKX2-1-AS1 knockdown determined by microarray analysis, 48 h after treatment (n = 6). ( C ) qPCR validation of down-regulated and up-regulated genes in NKX2-1-AS1 knockdown cells at 48 h after treatment (n = 3, *p

    Article Snippet: −1kbCD274-Luc (4ug) or 0-Luc control were transfected in the presence of various concentrations of pCDNA3.1- NKX2-1-AS1 vector (0, 0.5, 1, 2.5, 3, 5, 7 ug) alone or in combination with the pCDNA3.1- NKX2-1 vector (2ug). pCMV-Green Renilla luciferase vector construct (ThermoFisher) was co-transfected to measure transfection efficiency.

    Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction

    NKX2-1-AS1 follows tissue-specific patterns of expression similar to NKX2-1 in human cells. ( A ) Expression of NKX2-1-AS1 and NKX2-1 determined by RT-PCR in lung cell lines. The NKX2-1-AS1 PCR fragments were sequenced to confirm the identity of the sequence. ( B ) Relative expression patterns of NKX2-1-AS1 and NKX2-1 in tissues and cell lines, including normal human adult lung and thyroid and H441 and H661 cell lines, as determined by qPCR (n = 3; *p

    Journal: Scientific Reports

    Article Title: NKX2-1-AS1 negatively regulates CD274/PD-L1, cell-cell interaction genes, and limits human lung carcinoma cell migration

    doi: 10.1038/s41598-018-32793-5

    Figure Lengend Snippet: NKX2-1-AS1 follows tissue-specific patterns of expression similar to NKX2-1 in human cells. ( A ) Expression of NKX2-1-AS1 and NKX2-1 determined by RT-PCR in lung cell lines. The NKX2-1-AS1 PCR fragments were sequenced to confirm the identity of the sequence. ( B ) Relative expression patterns of NKX2-1-AS1 and NKX2-1 in tissues and cell lines, including normal human adult lung and thyroid and H441 and H661 cell lines, as determined by qPCR (n = 3; *p

    Article Snippet: −1kbCD274-Luc (4ug) or 0-Luc control were transfected in the presence of various concentrations of pCDNA3.1- NKX2-1-AS1 vector (0, 0.5, 1, 2.5, 3, 5, 7 ug) alone or in combination with the pCDNA3.1- NKX2-1 vector (2ug). pCMV-Green Renilla luciferase vector construct (ThermoFisher) was co-transfected to measure transfection efficiency.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Real-time Polymerase Chain Reaction

    NKX2-1-AS1 knockdown does not affect proliferation or apoptosis of H441 cells. ( A ) Cell growth was determined by counting cells at 24, 48 and 72 h after treatment with a pool of 3 siRNAs targeting NKX2-1-AS1 (n = 3). ( B ) Analysis of cell cycle stage by measuring DNA cell content by flow cytometry in NKX2-1-AS1 knockdown cells compared to non-silencing control at 48 h after treatment (n = 3). No significant change in cell number in each cell cycle stage was observed in NKX2-1-AS1 knockdown cells compared to non-silencing control. ( C ) No significant change in apoptosis was observed in NKX2-1-AS1 knockdown cells compared to non-silencing control as measured by annexin-V binding.

    Journal: Scientific Reports

    Article Title: NKX2-1-AS1 negatively regulates CD274/PD-L1, cell-cell interaction genes, and limits human lung carcinoma cell migration

    doi: 10.1038/s41598-018-32793-5

    Figure Lengend Snippet: NKX2-1-AS1 knockdown does not affect proliferation or apoptosis of H441 cells. ( A ) Cell growth was determined by counting cells at 24, 48 and 72 h after treatment with a pool of 3 siRNAs targeting NKX2-1-AS1 (n = 3). ( B ) Analysis of cell cycle stage by measuring DNA cell content by flow cytometry in NKX2-1-AS1 knockdown cells compared to non-silencing control at 48 h after treatment (n = 3). No significant change in cell number in each cell cycle stage was observed in NKX2-1-AS1 knockdown cells compared to non-silencing control. ( C ) No significant change in apoptosis was observed in NKX2-1-AS1 knockdown cells compared to non-silencing control as measured by annexin-V binding.

    Article Snippet: −1kbCD274-Luc (4ug) or 0-Luc control were transfected in the presence of various concentrations of pCDNA3.1- NKX2-1-AS1 vector (0, 0.5, 1, 2.5, 3, 5, 7 ug) alone or in combination with the pCDNA3.1- NKX2-1 vector (2ug). pCMV-Green Renilla luciferase vector construct (ThermoFisher) was co-transfected to measure transfection efficiency.

    Techniques: Flow Cytometry, Cytometry, Binding Assay

    NKX2-1-AS1 does not regulate expression of genes in the 14q13.3 chromosomal region. ( A ) Scheme of human chr14 within the 14q13.3 cytoband region indicating selected genes neighboring NKX2-1-AS1 . ( B ) qPCR analyses of NKX2-1-AS1 in H441 cells treated with a pool of three siRNAs targeting NKX2-1-AS1 exon 2 show significant down-regulation of NKX2-1-AS1 at 48 h and 72 h post transfection (n = 6; **p = 0.01 and ***p = 0.005 respectively). ( C ) qPCR analyses of the neighboring protein coding gene NKX2-1 in NKX2-1-AS1 knocked-down cells show no changes in NKX2-1 RNA levels (n = 6). ( D ) Representative western blots of NKX2-1 protein in non-silencing control [c] and NKX2-1-AS1 siRNA [si] treated H441 cells normalized to β-actin. ( E ) Densitometry of the western blot signals normalized to β-actin indicates that NKX2-1 protein levels also remained unchanged; n = 3. ( F ) Expression levels of NKX2-1-AS1 in the knockdown cells at 48 h and of other genes in the 14q13.3 chromosomal region as determined by microarray analysis; n = 6. ( G ) qPCR analysis in the NKX2-1-AS1 knockdown cells at 24, 48 and 72 h of MBIP ; (H) NKX2-8 ; and (I) PAX9 . n = 6, *p = 0.002.

    Journal: Scientific Reports

    Article Title: NKX2-1-AS1 negatively regulates CD274/PD-L1, cell-cell interaction genes, and limits human lung carcinoma cell migration

    doi: 10.1038/s41598-018-32793-5

    Figure Lengend Snippet: NKX2-1-AS1 does not regulate expression of genes in the 14q13.3 chromosomal region. ( A ) Scheme of human chr14 within the 14q13.3 cytoband region indicating selected genes neighboring NKX2-1-AS1 . ( B ) qPCR analyses of NKX2-1-AS1 in H441 cells treated with a pool of three siRNAs targeting NKX2-1-AS1 exon 2 show significant down-regulation of NKX2-1-AS1 at 48 h and 72 h post transfection (n = 6; **p = 0.01 and ***p = 0.005 respectively). ( C ) qPCR analyses of the neighboring protein coding gene NKX2-1 in NKX2-1-AS1 knocked-down cells show no changes in NKX2-1 RNA levels (n = 6). ( D ) Representative western blots of NKX2-1 protein in non-silencing control [c] and NKX2-1-AS1 siRNA [si] treated H441 cells normalized to β-actin. ( E ) Densitometry of the western blot signals normalized to β-actin indicates that NKX2-1 protein levels also remained unchanged; n = 3. ( F ) Expression levels of NKX2-1-AS1 in the knockdown cells at 48 h and of other genes in the 14q13.3 chromosomal region as determined by microarray analysis; n = 6. ( G ) qPCR analysis in the NKX2-1-AS1 knockdown cells at 24, 48 and 72 h of MBIP ; (H) NKX2-8 ; and (I) PAX9 . n = 6, *p = 0.002.

    Article Snippet: −1kbCD274-Luc (4ug) or 0-Luc control were transfected in the presence of various concentrations of pCDNA3.1- NKX2-1-AS1 vector (0, 0.5, 1, 2.5, 3, 5, 7 ug) alone or in combination with the pCDNA3.1- NKX2-1 vector (2ug). pCMV-Green Renilla luciferase vector construct (ThermoFisher) was co-transfected to measure transfection efficiency.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Microarray

    NKX2-1-AS1 inhibits cell motility in H441 cells. ( A ) Representative wound healing analysis of H441 cells treated with NKX2-1-AS1 siRNAs or non-silencing siRNA control. Cells were treated with the siRNAs for 24 h before the scratch was performed (0 h). Three images per scratch were taken at 0, 24, 48 and 72 h in 3 independent experiments. ( B ) Average wound area closed determined in the above images (*p

    Journal: Scientific Reports

    Article Title: NKX2-1-AS1 negatively regulates CD274/PD-L1, cell-cell interaction genes, and limits human lung carcinoma cell migration

    doi: 10.1038/s41598-018-32793-5

    Figure Lengend Snippet: NKX2-1-AS1 inhibits cell motility in H441 cells. ( A ) Representative wound healing analysis of H441 cells treated with NKX2-1-AS1 siRNAs or non-silencing siRNA control. Cells were treated with the siRNAs for 24 h before the scratch was performed (0 h). Three images per scratch were taken at 0, 24, 48 and 72 h in 3 independent experiments. ( B ) Average wound area closed determined in the above images (*p

    Article Snippet: −1kbCD274-Luc (4ug) or 0-Luc control were transfected in the presence of various concentrations of pCDNA3.1- NKX2-1-AS1 vector (0, 0.5, 1, 2.5, 3, 5, 7 ug) alone or in combination with the pCDNA3.1- NKX2-1 vector (2ug). pCMV-Green Renilla luciferase vector construct (ThermoFisher) was co-transfected to measure transfection efficiency.

    Techniques:

    NKX2-1-AS1 overexpression reduces CD274 expression levels in A549 cells in part by impairing NKX2-1 protein binding to the CD274 promoter. ChIP-qPCR analysis of NKX2-1 protein binding to ( A ) CD274 promoter (n = 4; p = 0.005), ( B ) CLDN1 promoter (n = 5), and ( C ) PTPN1 promoter (n = 5) in H441 cells transfected with NKX2-1-AS1 siRNAs or non-silencing control. ( D ) NKX2-1 co-transfection with the −1kb CD274 -Luc vector results in higher luciferase activity (3-fold in the absence of NKX2-1-AS1 , 0ug). NKX2-1-AS1 overexpression reduces the activity of the −1kb CD274 promoter in a dose-dependent manner both in the absence ( E ) (n = 3-4; ANOVA p = 0.003) or presence ( F ) (n = 3-4; ANOVA p = 0.0001) of NKX2-1 overexpression. NKX2-1-AS1 overexpression reduces the expression of the endogenous CD274 gene in a dose-dependent manner both in the absence ( G ) (n = 3-4; ANOVA p = 0.001) or presence ( H ) (n = 3-4; ANOVA p = 0.05) of NKX2-1 overexpression. (I) RIP-qPCR analysis of NKX2-1-AS1 pull down by NKX2-1 antibody compared to IgG control (n = 6; p = 0.05).

    Journal: Scientific Reports

    Article Title: NKX2-1-AS1 negatively regulates CD274/PD-L1, cell-cell interaction genes, and limits human lung carcinoma cell migration

    doi: 10.1038/s41598-018-32793-5

    Figure Lengend Snippet: NKX2-1-AS1 overexpression reduces CD274 expression levels in A549 cells in part by impairing NKX2-1 protein binding to the CD274 promoter. ChIP-qPCR analysis of NKX2-1 protein binding to ( A ) CD274 promoter (n = 4; p = 0.005), ( B ) CLDN1 promoter (n = 5), and ( C ) PTPN1 promoter (n = 5) in H441 cells transfected with NKX2-1-AS1 siRNAs or non-silencing control. ( D ) NKX2-1 co-transfection with the −1kb CD274 -Luc vector results in higher luciferase activity (3-fold in the absence of NKX2-1-AS1 , 0ug). NKX2-1-AS1 overexpression reduces the activity of the −1kb CD274 promoter in a dose-dependent manner both in the absence ( E ) (n = 3-4; ANOVA p = 0.003) or presence ( F ) (n = 3-4; ANOVA p = 0.0001) of NKX2-1 overexpression. NKX2-1-AS1 overexpression reduces the expression of the endogenous CD274 gene in a dose-dependent manner both in the absence ( G ) (n = 3-4; ANOVA p = 0.001) or presence ( H ) (n = 3-4; ANOVA p = 0.05) of NKX2-1 overexpression. (I) RIP-qPCR analysis of NKX2-1-AS1 pull down by NKX2-1 antibody compared to IgG control (n = 6; p = 0.05).

    Article Snippet: −1kbCD274-Luc (4ug) or 0-Luc control were transfected in the presence of various concentrations of pCDNA3.1- NKX2-1-AS1 vector (0, 0.5, 1, 2.5, 3, 5, 7 ug) alone or in combination with the pCDNA3.1- NKX2-1 vector (2ug). pCMV-Green Renilla luciferase vector construct (ThermoFisher) was co-transfected to measure transfection efficiency.

    Techniques: Over Expression, Expressing, Protein Binding, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Cotransfection, Plasmid Preparation, Luciferase, Activity Assay