mammalian expression vector pcdna 3.1 Thermo Fisher Search Results


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  • 90
    Thermo Fisher pcdna3 mammalian expression vector
    Ape1/Ref-1 increases GFRα1 promoter activity by enhancing p50 NF-κB activation. (A) The upper panel shows a schematic representation of the human GFRα1 promoter region. The three putative NF-κB-binding sites spanning positions −349 to −335 (NF1), −300 to −287 (NF2), and −155 to −143 (NF3) are shown in green. The middle and lower panels show the GFRα1 promoter region of the promoter-reporter constructs p2284 (positions −2291 to −7), p964 (positions −2291 to −1327), and p509 (positions −575 to −66). (B) Parent cells and <t>pcDNA3-</t> and Ape1/Ref-11-expressing cells were transfected with pGL3-Basic, p2284, p964, or p509. Representations of the promoters are shown. The values reported are means ± the SD from six separate experiments. **, P
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    Thermo Fisher clones mammalian expression vector pcdna3 1
    HOTAIR expression correlates positively with HPV16 E7 expression. ( a – c ) Correlation analysis between HOTAIR and HPV16 E7 expression among CaCx cases, episomal and integrated CaCx cases, respectively. ( d , e ) Box plots representing distribution of HPV16 E7 and HOTAIR expression levels respectively, in C33A cell line at various time points post-transfection (p. t.) of <t>pcDNA3.1-HPV16</t> E7 vector. ( f , g ) Box plots representing distribution of expression levels of HPV16 E7 and HOTAIR among SiHa and Caski cell lines respectively.
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    Thermo Fisher mammalian expression vector pcdna3
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher mammalian expression vector pcdna3 1a
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher pcdna3 1hygro expression vector
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher mammalian expression vector pcdna3 1v5histopo pcdna3 1vh
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher pcdna3 1 nv5 dest mammalian expression vector
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher pcdna3 1 his a
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher pcdna3 1 myc his mammalian expression vector
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher pcdna3 1a myc his mammalian expression vector
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher pcdna3 hygro mammalian expression vector
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher pcdna3 1 v5 his a
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher mammalian expression vector pcdna3 1d v5 his topo
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher pcdna3 1d topo mammalian expression vector
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher mammalian expression vector pcdna3 1 v5 his
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher gateway converted pcdna3 1 mammalian expression vector
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher pcdna3 1 vector
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher ecor1 xhoi digested mammalian expression vector pcdna3 1 myc hisa
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher pcdna5 frt mammalian expression vector
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher mammalian expression plasmid pc3 1dna
    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with <t>pcDNA3.1/Zeo(+)</t> (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.
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    Thermo Fisher pcdna3 1 directional topo expression kit
    GSK3ß overexpression and S9A modification regulates ERK phosphorylation in primary rat PASMCs. ( A ) Primary rat PASMCs were transiently transfected with GSK3ß wild type (GSK3ß WT), constitutively active GSK3ß (GSK3ß S9A), empty vector <t>(pcDNA3.1</t> <t>TOPO).</t> 24 hrs post transfection, expression and phosphorylation of GSK3ß and ERK were analyzed by western blotting followed by ( B ) densitometric quantification. All values are expressed as mean ± SEM (n = 4). Values were presented significant as ***P
    Pcdna3 1 Directional Topo Expression Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 452 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mammalian expression vector pcdna3 1zeo
    GSK3ß overexpression and S9A modification regulates ERK phosphorylation in primary rat PASMCs. ( A ) Primary rat PASMCs were transiently transfected with GSK3ß wild type (GSK3ß WT), constitutively active GSK3ß (GSK3ß S9A), empty vector <t>(pcDNA3.1</t> <t>TOPO).</t> 24 hrs post transfection, expression and phosphorylation of GSK3ß and ERK were analyzed by western blotting followed by ( B ) densitometric quantification. All values are expressed as mean ± SEM (n = 4). Values were presented significant as ***P
    Mammalian Expression Vector Pcdna3 1zeo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bicistronic mammalian expression vector pcdna3 1
    GSK3ß overexpression and S9A modification regulates ERK phosphorylation in primary rat PASMCs. ( A ) Primary rat PASMCs were transiently transfected with GSK3ß wild type (GSK3ß WT), constitutively active GSK3ß (GSK3ß S9A), empty vector <t>(pcDNA3.1</t> <t>TOPO).</t> 24 hrs post transfection, expression and phosphorylation of GSK3ß and ERK were analyzed by western blotting followed by ( B ) densitometric quantification. All values are expressed as mean ± SEM (n = 4). Values were presented significant as ***P
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    Thermo Fisher mammalian expression vector pcdna3 1 v5his topo
    GSK3ß overexpression and S9A modification regulates ERK phosphorylation in primary rat PASMCs. ( A ) Primary rat PASMCs were transiently transfected with GSK3ß wild type (GSK3ß WT), constitutively active GSK3ß (GSK3ß S9A), empty vector <t>(pcDNA3.1</t> <t>TOPO).</t> 24 hrs post transfection, expression and phosphorylation of GSK3ß and ERK were analyzed by western blotting followed by ( B ) densitometric quantification. All values are expressed as mean ± SEM (n = 4). Values were presented significant as ***P
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    Thermo Fisher mammalian expression plasmid topo pcdna3 1 his v5
    GSK3ß overexpression and S9A modification regulates ERK phosphorylation in primary rat PASMCs. ( A ) Primary rat PASMCs were transiently transfected with GSK3ß wild type (GSK3ß WT), constitutively active GSK3ß (GSK3ß S9A), empty vector <t>(pcDNA3.1</t> <t>TOPO).</t> 24 hrs post transfection, expression and phosphorylation of GSK3ß and ERK were analyzed by western blotting followed by ( B ) densitometric quantification. All values are expressed as mean ± SEM (n = 4). Values were presented significant as ***P
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    Thermo Fisher pcdna3 1 v5 his topo ta expression kit
    AID and Ung are critical for the generation of staggered DSBs and CSR. A , Forced expression of Aid results in occurrence of staggered DSBs. Paired samples of low Aid expression 4D11 B cells were transfected with an empty <t>pcDNA3.1</t> vector or a pcDNA3.1 vector containing human Aid cDNA, and then selected in a 21-day culture in the presence of G418. The selected cells were further stimulated for three days with agonistic anti-huCD40 mAb and huIL-4, and harvested to prepare genomic DNA and RNA. To analyze DSBs, blunt or total (blunt plus staggered) Sμ upstream DSB ends were amplified by LM-PCR after pre-treatment of genomic DNA with nil (T4−) or T4 pol (T4+) before being ligated with BW linker. Linker-ligated genomic DNA (8 ng from 1280 B cells) was serially two-fold diluted into unligated homologous genomic DNA and used as template in LM-PCR (lanes 1–8 in each panel). Germline Iμ-Cμ, Iγ1-Cγ1, circle Iγ1-Cμ, mature V H DJ H -Cμ1, total Aid or expression vector-encoded Aid (vector Aid ), and β- actin transcripts were analyzed by RT-PCR using serially two-fold diluted cDNA as templates. B , Expression of DN Aid abrogates the generation of staggered DNA ends. Paired samples of spontaneously switching 4B6 B cells transfected with an empty pcDNA3.1 vector or a pcDNA3.1 vector containing a human DN Aid construct and then selected in a 21 days culture in the presence of G418. The selected cells were further cultured with nil for three days and then harvested to prepare genomic DNA and RNA. To analyze DSBs, blunt or total (blunt plus staggered) Sμ upstream DSB ends were amplified by LM-PCR after pre-treatment of genomic DNA with nil (T4−) or T4 pol (T4+) before being ligated with BW linker. Linker-ligated genomic DNA (8 ng from 1280 B cells) was serially two-fold diluted into unligated homologous genomic DNA and used as template in LM-PCR (lanes 1–8 in each panel). Germline Iμ-Cμ, Iγ1-Cγ1, circle Iγ1-Cμ, mature V H DJ H -Cγ1, total Aid or expression vector-encoded DN Aid , and β- actin transcripts were analyzed by RT-PCR using serially two-fold diluted cDNA as templates. C , Expression of Ugi abrogates the emergence of staggered DSBs. Paired samples of spontaneously switching human 4B6 B cells or CSR-inducible human 3G10 B cells were transfected with an empty pcDNA3.1 vector or a pcDNA3.1 vector containing an Ugi construct and selected in a 21 days culture in the presence of G418. Genomic DNA, RNA and whole-cell extract were prepared from transfected 4B6 B cells cultured with nil for three days and transfected 3G10 B cells stimulated for three day with nil or agonistic anti-huCD40 mAb and huIL-4. To analyze DSBs, blunt or total (blunt plus staggered) Sμ upstream DSB ends were amplified by LM-PCR after pre-treatment of genomic DNA with nil (T4−) or T4 pol (T4+) before being ligated with BW linker. Linker-ligated genomic DNA (8 ng from 1280 B cells) was serially two-fold diluted into unligated homologous genomic DNA and used as template in LM-PCR (lanes 1–8 in each panel). Ung activity of the B cells was detected by incubate 10 μg, 1.0 μg or 0.1 μg (protein) of clarified whole-cell extract with a [ 32 P] labeled double-stranded oligonucleotide containing a single dU/dG residue. The reaction products were resolved in 15% TBE-urea polyacrylamide gels. Germline Iμ-Cμ, Iγ1-Cγ1, circle Iγ1-Cμ, mature V H DJ H -Cγ1, Aid and β- actin transcripts were analyzed by RT-PCR using serially two-fold diluted cDNA as templates. D , Intra-S region recombination occurs in the absence of AID or Ung. DNA was PCR-amplified from a mouse germline S□ region, as inserted into pCR-Blunt <t>II-TOPO®</t> plasmid (left panel) or genomic DNA isolated from aicda +/+ and aicda −/− (center panel) or ung +/+ and ung −/− (right panel) mouse B cells stimulated by LPS and moIL-4 for three days (left, center and right panels were from different gels), and specified by Southern-blot using a specific [ 32 P]-labeled Sμ probe. Recombined intra-Sμ DNA resulted in Sμ DNA amplification products smaller than those expected based on germline Sμ (3730 bp) region length (closed arrowhead). Migration of molecular weight markers is indicated by open arrowheads.
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    Long-term exposure of kisspeptin inhibits insulin secretion in NIT-1 cells. ( a ) NIT-1 cells were transfected with or without <t>pcDNA3.1</t> + mKiss1-T2A-GFP plasmid, then fixed and stained with Hoechst 33342. Nucleus and GFP signal are shown in blue and green, respectively. The overlay of signals was processed by ImageJ. ( b ) Representative blots of GFP and mouse kisspeptin from transfected NIT-1 cells are shown. ( c ) Insulin secretion ability of control and transfected NIT-1 cells were determined by the amount of secreted luciferase under basal and glucose-stimulated condition. The relative luciferase activity was normalized by total protein in cell lysates. Data represent the means ± standard errors of the mean (n = 3). *Compared with the basal level in the control group; ** p
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    Long-term exposure of kisspeptin inhibits insulin secretion in NIT-1 cells. ( a ) NIT-1 cells were transfected with or without <t>pcDNA3.1</t> + mKiss1-T2A-GFP plasmid, then fixed and stained with Hoechst 33342. Nucleus and GFP signal are shown in blue and green, respectively. The overlay of signals was processed by ImageJ. ( b ) Representative blots of GFP and mouse kisspeptin from transfected NIT-1 cells are shown. ( c ) Insulin secretion ability of control and transfected NIT-1 cells were determined by the amount of secreted luciferase under basal and glucose-stimulated condition. The relative luciferase activity was normalized by total protein in cell lysates. Data represent the means ± standard errors of the mean (n = 3). *Compared with the basal level in the control group; ** p
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    Long-term exposure of kisspeptin inhibits insulin secretion in NIT-1 cells. ( a ) NIT-1 cells were transfected with or without <t>pcDNA3.1</t> + mKiss1-T2A-GFP plasmid, then fixed and stained with Hoechst 33342. Nucleus and GFP signal are shown in blue and green, respectively. The overlay of signals was processed by ImageJ. ( b ) Representative blots of GFP and mouse kisspeptin from transfected NIT-1 cells are shown. ( c ) Insulin secretion ability of control and transfected NIT-1 cells were determined by the amount of secreted luciferase under basal and glucose-stimulated condition. The relative luciferase activity was normalized by total protein in cell lysates. Data represent the means ± standard errors of the mean (n = 3). *Compared with the basal level in the control group; ** p
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    Long-term exposure of kisspeptin inhibits insulin secretion in NIT-1 cells. ( a ) NIT-1 cells were transfected with or without <t>pcDNA3.1</t> + mKiss1-T2A-GFP plasmid, then fixed and stained with Hoechst 33342. Nucleus and GFP signal are shown in blue and green, respectively. The overlay of signals was processed by ImageJ. ( b ) Representative blots of GFP and mouse kisspeptin from transfected NIT-1 cells are shown. ( c ) Insulin secretion ability of control and transfected NIT-1 cells were determined by the amount of secreted luciferase under basal and glucose-stimulated condition. The relative luciferase activity was normalized by total protein in cell lysates. Data represent the means ± standard errors of the mean (n = 3). *Compared with the basal level in the control group; ** p
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    Thermo Fisher cytomegalovirus
    Long-term exposure of kisspeptin inhibits insulin secretion in NIT-1 cells. ( a ) NIT-1 cells were transfected with or without <t>pcDNA3.1</t> + mKiss1-T2A-GFP plasmid, then fixed and stained with Hoechst 33342. Nucleus and GFP signal are shown in blue and green, respectively. The overlay of signals was processed by ImageJ. ( b ) Representative blots of GFP and mouse kisspeptin from transfected NIT-1 cells are shown. ( c ) Insulin secretion ability of control and transfected NIT-1 cells were determined by the amount of secreted luciferase under basal and glucose-stimulated condition. The relative luciferase activity was normalized by total protein in cell lysates. Data represent the means ± standard errors of the mean (n = 3). *Compared with the basal level in the control group; ** p
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    Image Search Results


    Ape1/Ref-1 increases GFRα1 promoter activity by enhancing p50 NF-κB activation. (A) The upper panel shows a schematic representation of the human GFRα1 promoter region. The three putative NF-κB-binding sites spanning positions −349 to −335 (NF1), −300 to −287 (NF2), and −155 to −143 (NF3) are shown in green. The middle and lower panels show the GFRα1 promoter region of the promoter-reporter constructs p2284 (positions −2291 to −7), p964 (positions −2291 to −1327), and p509 (positions −575 to −66). (B) Parent cells and pcDNA3- and Ape1/Ref-11-expressing cells were transfected with pGL3-Basic, p2284, p964, or p509. Representations of the promoters are shown. The values reported are means ± the SD from six separate experiments. **, P

    Journal: Molecular and Cellular Biology

    Article Title: Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor ?1 Expression ▿Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor ?1 Expression ▿ ‡

    doi: 10.1128/MCB.01484-08

    Figure Lengend Snippet: Ape1/Ref-1 increases GFRα1 promoter activity by enhancing p50 NF-κB activation. (A) The upper panel shows a schematic representation of the human GFRα1 promoter region. The three putative NF-κB-binding sites spanning positions −349 to −335 (NF1), −300 to −287 (NF2), and −155 to −143 (NF3) are shown in green. The middle and lower panels show the GFRα1 promoter region of the promoter-reporter constructs p2284 (positions −2291 to −7), p964 (positions −2291 to −1327), and p509 (positions −575 to −66). (B) Parent cells and pcDNA3- and Ape1/Ref-11-expressing cells were transfected with pGL3-Basic, p2284, p964, or p509. Representations of the promoters are shown. The values reported are means ± the SD from six separate experiments. **, P

    Article Snippet: The amplified cDNA was then cloned into a pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA) and a pShuttle vector (Invitrogen).

    Techniques: Activity Assay, Activation Assay, Binding Assay, Construct, Expressing, Transfection

    GFRα1 expression after the adenovirus-mediated transfer of Ape1/Ref-1 in GM00637 cells. (A) The GM00637 cells were transfected with Ad-LacZ or Ad-Ape1/Ref-1 at a multiplicity of infection of 50, and the cells were harvested 48 h after the infection. The total RNA was extracted and subjected to semiquantitative RT-PCR using the Ape1/Ref-1 -, GFRα -, and GAPDH -specific primers. (B) Protein extracts prepared 48 h after the infection with Ad-LacZ or Ad-Ape1/Ref-1. A 20-μg portion of the total protein was loaded onto an SDS-polyacrylamide gel for Western blot analysis. Antibodies to Ape1/Ref-1 and GFRα1 were used. The detection of α-tubulin was used as the loading control. (C) The GM00637 cells were transfected with control pcDNA3 vector (control), repair Ape1/Ref-1 mutant expression vector (ΔRD), or redox Ape1/Ref-1 mutant expression vector (ΔAPD), and the total RNA was then extracted and subjected to semiquantitative RT-PCR using the Redox Ape1/Ref-1 , Repair Ape1/Ref-1 , GFRα, and GAPDH primers.

    Journal: Molecular and Cellular Biology

    Article Title: Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor ?1 Expression ▿Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor ?1 Expression ▿ ‡

    doi: 10.1128/MCB.01484-08

    Figure Lengend Snippet: GFRα1 expression after the adenovirus-mediated transfer of Ape1/Ref-1 in GM00637 cells. (A) The GM00637 cells were transfected with Ad-LacZ or Ad-Ape1/Ref-1 at a multiplicity of infection of 50, and the cells were harvested 48 h after the infection. The total RNA was extracted and subjected to semiquantitative RT-PCR using the Ape1/Ref-1 -, GFRα -, and GAPDH -specific primers. (B) Protein extracts prepared 48 h after the infection with Ad-LacZ or Ad-Ape1/Ref-1. A 20-μg portion of the total protein was loaded onto an SDS-polyacrylamide gel for Western blot analysis. Antibodies to Ape1/Ref-1 and GFRα1 were used. The detection of α-tubulin was used as the loading control. (C) The GM00637 cells were transfected with control pcDNA3 vector (control), repair Ape1/Ref-1 mutant expression vector (ΔRD), or redox Ape1/Ref-1 mutant expression vector (ΔAPD), and the total RNA was then extracted and subjected to semiquantitative RT-PCR using the Redox Ape1/Ref-1 , Repair Ape1/Ref-1 , GFRα, and GAPDH primers.

    Article Snippet: The amplified cDNA was then cloned into a pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA) and a pShuttle vector (Invitrogen).

    Techniques: Expressing, Transfection, Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Mutagenesis

    Effect of CR749391 overexpression on tumor growth in vivo . A xenograft model derived from BGC-823 cells transfected with pcDNA3.1/CR749391 or empty vector was established. (A) CR749391 expression in tissues of resected tumors was determined by reverse transcription-quantitative polymerase chain reaction. (B) Body weight curves of the mice and (C) tumor volume over the course of the experiment. Values are expressed as the mean ± standard deviation (n=4). *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: LncRNA CR749391 acts as a tumor suppressor to upregulate KLF6 expression via interacting with miR-181a in gastric cancer

    doi: 10.3892/etm.2019.8226

    Figure Lengend Snippet: Effect of CR749391 overexpression on tumor growth in vivo . A xenograft model derived from BGC-823 cells transfected with pcDNA3.1/CR749391 or empty vector was established. (A) CR749391 expression in tissues of resected tumors was determined by reverse transcription-quantitative polymerase chain reaction. (B) Body weight curves of the mice and (C) tumor volume over the course of the experiment. Values are expressed as the mean ± standard deviation (n=4). *P

    Article Snippet: The full length of CR749391 were then amplified from human complementary (c)DNA using polymerase chain reaction (PCR) and subcloned into the pcDNA3.1 mammalian expression vector (Invitrogen; Thermo Fisher Scientific, Inc.).

    Techniques: Over Expression, In Vivo, Derivative Assay, Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Standard Deviation

    Effect of CR749391 on gastric cancer cell migration and invasion in vitro . Transwell assays were performed to investigate changes in cell migration and invasion. (A and B) GES-1 cells were transfected with si-CR749391 or si-NC, and (C and D) BGC-823 cells were transfected with pcDNA3.1/CR749391 vector or empty vector control, and then subjected to Transwell assays. Magnification, ×100. The data are expressed as the mean ± standard deviation of three independent experiments. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: LncRNA CR749391 acts as a tumor suppressor to upregulate KLF6 expression via interacting with miR-181a in gastric cancer

    doi: 10.3892/etm.2019.8226

    Figure Lengend Snippet: Effect of CR749391 on gastric cancer cell migration and invasion in vitro . Transwell assays were performed to investigate changes in cell migration and invasion. (A and B) GES-1 cells were transfected with si-CR749391 or si-NC, and (C and D) BGC-823 cells were transfected with pcDNA3.1/CR749391 vector or empty vector control, and then subjected to Transwell assays. Magnification, ×100. The data are expressed as the mean ± standard deviation of three independent experiments. *P

    Article Snippet: The full length of CR749391 were then amplified from human complementary (c)DNA using polymerase chain reaction (PCR) and subcloned into the pcDNA3.1 mammalian expression vector (Invitrogen; Thermo Fisher Scientific, Inc.).

    Techniques: Migration, In Vitro, Transfection, Plasmid Preparation, Standard Deviation

    CR749391 expression in gastric cancer cells and its effect on cell proliferation in vitro . (A and B) MTT assays were performed to determine the proliferation of (A) si-CR749391-transfected GES-1 cells or (B) pcDNA3.1/CR749391-transfected BGC-823 cells. (C-F) The apoptotic rates of (C and D) GES-1 cells transfected with si-CR749391 and (E and F) BGC-823 cells transfected with pcDNA3.1/CR749391 vector were detected by flow cytometry. Values are expressed as the mean ± standard deviation from three independent experiments. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: LncRNA CR749391 acts as a tumor suppressor to upregulate KLF6 expression via interacting with miR-181a in gastric cancer

    doi: 10.3892/etm.2019.8226

    Figure Lengend Snippet: CR749391 expression in gastric cancer cells and its effect on cell proliferation in vitro . (A and B) MTT assays were performed to determine the proliferation of (A) si-CR749391-transfected GES-1 cells or (B) pcDNA3.1/CR749391-transfected BGC-823 cells. (C-F) The apoptotic rates of (C and D) GES-1 cells transfected with si-CR749391 and (E and F) BGC-823 cells transfected with pcDNA3.1/CR749391 vector were detected by flow cytometry. Values are expressed as the mean ± standard deviation from three independent experiments. *P

    Article Snippet: The full length of CR749391 were then amplified from human complementary (c)DNA using polymerase chain reaction (PCR) and subcloned into the pcDNA3.1 mammalian expression vector (Invitrogen; Thermo Fisher Scientific, Inc.).

    Techniques: Expressing, In Vitro, MTT Assay, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Standard Deviation

    Functional analysis of LINC00703. BGC-823 cells were transfected with pcDNA3.1 or LINC00703 overexpressing vector (LINC00703). (A and B) Measurements of cell proliferation using MTT assay (A) and PCNA protein level (B). (C) Flow cytometry analysis of cell apoptotic rates. (D) Results of transwell assays reflecting the changes in cell migration and invasiveness (100×). All data are presented as means±standard deviation (n=3, biological replicates). LINC00703 = long noncoding RNA 00703; MTT = 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCNA = proliferating cell nuclear antigen. * P

    Journal: Journal of Gastric Cancer

    Article Title: LINC00703 Acts as a Tumor Suppressor via Regulating miR-181a/KLF6 Axis in Gastric Cancer

    doi: 10.5230/jgc.2019.19.e43

    Figure Lengend Snippet: Functional analysis of LINC00703. BGC-823 cells were transfected with pcDNA3.1 or LINC00703 overexpressing vector (LINC00703). (A and B) Measurements of cell proliferation using MTT assay (A) and PCNA protein level (B). (C) Flow cytometry analysis of cell apoptotic rates. (D) Results of transwell assays reflecting the changes in cell migration and invasiveness (100×). All data are presented as means±standard deviation (n=3, biological replicates). LINC00703 = long noncoding RNA 00703; MTT = 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCNA = proliferating cell nuclear antigen. * P

    Article Snippet: Vectors construction and luciferase reporter activity assay The full length of LINC00703 was amplified using cDNA as a template and then subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen).

    Techniques: Functional Assay, Transfection, Plasmid Preparation, MTT Assay, Flow Cytometry, Cytometry, Migration, Standard Deviation

    LINC00703 regulates KLF6 expression. (A) The qRT-PCR analysis, demonstrating LINC00703 expression in several gastric cancer cell lines (MGC-803, SGC-7901, and BGC-823), compared with normal gastric epithelium cell line (GES-1). (B) LINC00703 expression analysis in BGC-823 cells, transfected with pcDNA3.1 or LINC00703-overexpressing vector (LINC00703). (C and D) Expression levels of miR-181a and KLF6 after 48 hours, following the transfection of pcDNA3.1 or LINC00703 in BGC-823 cells. (E) The KLF6 protein level in BGC-823 cells, transfected with miR-181a mimic, LINC00703 or both miR-181a mimic and LINC00703. All data are presented as means±standard deviation (n=5, biological replicates). LINC00703 = long noncoding RNA 00703; KLF = Kruppel-like factor; qRT-PCR = quantitative real-time polymerase chain reaction. * P

    Journal: Journal of Gastric Cancer

    Article Title: LINC00703 Acts as a Tumor Suppressor via Regulating miR-181a/KLF6 Axis in Gastric Cancer

    doi: 10.5230/jgc.2019.19.e43

    Figure Lengend Snippet: LINC00703 regulates KLF6 expression. (A) The qRT-PCR analysis, demonstrating LINC00703 expression in several gastric cancer cell lines (MGC-803, SGC-7901, and BGC-823), compared with normal gastric epithelium cell line (GES-1). (B) LINC00703 expression analysis in BGC-823 cells, transfected with pcDNA3.1 or LINC00703-overexpressing vector (LINC00703). (C and D) Expression levels of miR-181a and KLF6 after 48 hours, following the transfection of pcDNA3.1 or LINC00703 in BGC-823 cells. (E) The KLF6 protein level in BGC-823 cells, transfected with miR-181a mimic, LINC00703 or both miR-181a mimic and LINC00703. All data are presented as means±standard deviation (n=5, biological replicates). LINC00703 = long noncoding RNA 00703; KLF = Kruppel-like factor; qRT-PCR = quantitative real-time polymerase chain reaction. * P

    Article Snippet: Vectors construction and luciferase reporter activity assay The full length of LINC00703 was amplified using cDNA as a template and then subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen).

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Standard Deviation, Real-time Polymerase Chain Reaction

    Effect of LINC00703 on tumor growth in vivo. (A) Tumors were subcutaneously induced in nude mice. BGC-832 cells that were stably transfected with pcDNA3.1 or LINC00703 overexpressing vectors (LINC00703) were injected into the flanks of nude mice for 12 days. (B and C) Dynamics of tumor volume (A) and mice body weight (B) according to the measurements every 3 days. (D) The tumors were dissected and weighed at the end of experiment. (E) qRT-PCR analysis of the expression of LINC00703, miR-181a and KLF6 in resected tumor tissues from the nude mice. All data are presented as means±standard deviation (n=8, for each group). LINC00703 = long noncoding RNA 00703; qRT-PCR = quantitative real-time polymerase chain reaction; KLF = Kruppel-like factor. * P

    Journal: Journal of Gastric Cancer

    Article Title: LINC00703 Acts as a Tumor Suppressor via Regulating miR-181a/KLF6 Axis in Gastric Cancer

    doi: 10.5230/jgc.2019.19.e43

    Figure Lengend Snippet: Effect of LINC00703 on tumor growth in vivo. (A) Tumors were subcutaneously induced in nude mice. BGC-832 cells that were stably transfected with pcDNA3.1 or LINC00703 overexpressing vectors (LINC00703) were injected into the flanks of nude mice for 12 days. (B and C) Dynamics of tumor volume (A) and mice body weight (B) according to the measurements every 3 days. (D) The tumors were dissected and weighed at the end of experiment. (E) qRT-PCR analysis of the expression of LINC00703, miR-181a and KLF6 in resected tumor tissues from the nude mice. All data are presented as means±standard deviation (n=8, for each group). LINC00703 = long noncoding RNA 00703; qRT-PCR = quantitative real-time polymerase chain reaction; KLF = Kruppel-like factor. * P

    Article Snippet: Vectors construction and luciferase reporter activity assay The full length of LINC00703 was amplified using cDNA as a template and then subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen).

    Techniques: In Vivo, Mouse Assay, Stable Transfection, Transfection, Injection, Quantitative RT-PCR, Expressing, Standard Deviation, Real-time Polymerase Chain Reaction

    HOTAIR expression correlates positively with HPV16 E7 expression. ( a – c ) Correlation analysis between HOTAIR and HPV16 E7 expression among CaCx cases, episomal and integrated CaCx cases, respectively. ( d , e ) Box plots representing distribution of HPV16 E7 and HOTAIR expression levels respectively, in C33A cell line at various time points post-transfection (p. t.) of pcDNA3.1-HPV16 E7 vector. ( f , g ) Box plots representing distribution of expression levels of HPV16 E7 and HOTAIR among SiHa and Caski cell lines respectively.

    Journal: Scientific Reports

    Article Title: Bridging Links between Long Noncoding RNA HOTAIR and HPV Oncoprotein E7 in Cervical Cancer Pathogenesis

    doi: 10.1038/srep11724

    Figure Lengend Snippet: HOTAIR expression correlates positively with HPV16 E7 expression. ( a – c ) Correlation analysis between HOTAIR and HPV16 E7 expression among CaCx cases, episomal and integrated CaCx cases, respectively. ( d , e ) Box plots representing distribution of HPV16 E7 and HOTAIR expression levels respectively, in C33A cell line at various time points post-transfection (p. t.) of pcDNA3.1-HPV16 E7 vector. ( f , g ) Box plots representing distribution of expression levels of HPV16 E7 and HOTAIR among SiHa and Caski cell lines respectively.

    Article Snippet: Plasmids and clones Mammalian expression vector pcDNA3.1(+) was purchased from Invitrogen (Cat # V790-20) and used for cloning of HPV16 E7 ORF.

    Techniques: Expressing, Transfection, Plasmid Preparation

    Increased expression of PRC2-complex members, EZH2 and SUZ12, among CaCx cases appears to be HPV16 E7-dependent. ( a , b ) Box plots representing distribution of EZH2 and SUZ12 expression levels respectively, among different categories of cervical samples. ( c , d ) Box plots representing distribution of EZH2 and SUZ12 expression levels in C33A cell line, at various time points post-transfection (p.t.) of pcDNA3.1-HPV16 E7 vector.

    Journal: Scientific Reports

    Article Title: Bridging Links between Long Noncoding RNA HOTAIR and HPV Oncoprotein E7 in Cervical Cancer Pathogenesis

    doi: 10.1038/srep11724

    Figure Lengend Snippet: Increased expression of PRC2-complex members, EZH2 and SUZ12, among CaCx cases appears to be HPV16 E7-dependent. ( a , b ) Box plots representing distribution of EZH2 and SUZ12 expression levels respectively, among different categories of cervical samples. ( c , d ) Box plots representing distribution of EZH2 and SUZ12 expression levels in C33A cell line, at various time points post-transfection (p.t.) of pcDNA3.1-HPV16 E7 vector.

    Article Snippet: Plasmids and clones Mammalian expression vector pcDNA3.1(+) was purchased from Invitrogen (Cat # V790-20) and used for cloning of HPV16 E7 ORF.

    Techniques: Expressing, Transfection, Plasmid Preparation

    Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with pcDNA3.1/Zeo(+) (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Effect of the GPI anchor of human Thy-1 on antibody recognition and function

    doi: 10.1038/labinvest.2012.178

    Figure Lengend Snippet: Complete GPI anchor of Thy-1 required for recognition by monoclonal antibodies raised against membrane bound form (A) Mature Thy-1 detected by western blot in the CEs of NHLF and RFL-6 stably transfected with pcDNA3.1/Zeo(+) (pcDNA) or pcDNA3.1/Zeo (+) with a recombinant form of Thy-1 cloned in: WT THY1 (THY1), THY1 – GPI(DAF) (DAF), THY1 – GPI(TR3) (TR3), or FLAG – THY1 (FLAG). (B) Mature Thy-1 detected by western blot in CE containing FLAG – Thy-1 or CM containing PI-PLC released FLAG – Thy-1 or FLAG – sThy-1 using K117 and AS02. After stripping the membranes of antibodies for detecting mature Thy-1, FLAG was detected using M2.

    Article Snippet: Recombinant Constructs of THY1 For expression of wild type (WT) human Thy-1, the complete cDNA of human THY1 (AAH65559.1) was ligated into the mammalian expression vector pcDNA3.1/Zeo (+) (Invitrogen V860-20) with the Kozak sequence GCCGCC just upstream of the start codon.

    Techniques: Western Blot, Stable Transfection, Transfection, Recombinant, Clone Assay, Planar Chromatography, Stripping Membranes

    GSK3ß overexpression and S9A modification regulates ERK phosphorylation in primary rat PASMCs. ( A ) Primary rat PASMCs were transiently transfected with GSK3ß wild type (GSK3ß WT), constitutively active GSK3ß (GSK3ß S9A), empty vector (pcDNA3.1 TOPO). 24 hrs post transfection, expression and phosphorylation of GSK3ß and ERK were analyzed by western blotting followed by ( B ) densitometric quantification. All values are expressed as mean ± SEM (n = 4). Values were presented significant as ***P

    Journal: PLoS ONE

    Article Title: Glycogen Synthase Kinase 3beta Contributes to Proliferation of Arterial Smooth Muscle Cells in Pulmonary Hypertension

    doi: 10.1371/journal.pone.0018883

    Figure Lengend Snippet: GSK3ß overexpression and S9A modification regulates ERK phosphorylation in primary rat PASMCs. ( A ) Primary rat PASMCs were transiently transfected with GSK3ß wild type (GSK3ß WT), constitutively active GSK3ß (GSK3ß S9A), empty vector (pcDNA3.1 TOPO). 24 hrs post transfection, expression and phosphorylation of GSK3ß and ERK were analyzed by western blotting followed by ( B ) densitometric quantification. All values are expressed as mean ± SEM (n = 4). Values were presented significant as ***P

    Article Snippet: MiniPrep Plasmid Isolation Kit was obtained from PeqLab (Erlangen, Germany) and MaxiPrep Plasmid Isolation Kit, PCR purification Kit was purchased from QIAGEN (Hilden, Germany), pGEMT-Easy vector Kit was obtained from Promega, (Mannheim, Germany), pcDNA3.1 Directional TOPO Expression Kit, Lipofectamine 2000, T4 Ligase, Platinum Taq DNA Polymerase High Fidelity, Platinum SYBR Green qPCR SuperMix-UDG was purchased from Invitrogen (Karlsruhe, Germany), restriction enzymes (BamHI, BsmI, EcoRV) was purchased from New England BioLabs (Frankfurt, Germany).

    Techniques: Over Expression, Modification, Transfection, Plasmid Preparation, Expressing, Western Blot

    GSK3ß overexpression and S9A modification influences proliferation of primary rat PASMCs. ( A ) Primary rat PASMCs were transiently transfected with GSK3ß wild type (GSK3ß WT), constitutively active GSK3ß (GSK3ß S9A), empty vector (pcDNA3.1 TOPO). 24 hrs post transfection, expression and phosphorylation of GSK3ß were analyzed by western blotting. ( B ) Primary rat PASMCs transiently transfected with GSK3ß wild type (GSK3ß WT), constitutively active GSK3ß (GSK3ß S9A), empty vector (pcDNA3.1 TOPO) and proliferation was assessed after stimulation with 10% FCS for 24 hrs by [ 3 H]-thymidine incorporation. All values are expressed as mean ± SEM (n = 6, WT or S9A n = 12). Data were expressed as counts per minute (cpm) and normalized to the amount of cells per well. Values were presented significant as ***P

    Journal: PLoS ONE

    Article Title: Glycogen Synthase Kinase 3beta Contributes to Proliferation of Arterial Smooth Muscle Cells in Pulmonary Hypertension

    doi: 10.1371/journal.pone.0018883

    Figure Lengend Snippet: GSK3ß overexpression and S9A modification influences proliferation of primary rat PASMCs. ( A ) Primary rat PASMCs were transiently transfected with GSK3ß wild type (GSK3ß WT), constitutively active GSK3ß (GSK3ß S9A), empty vector (pcDNA3.1 TOPO). 24 hrs post transfection, expression and phosphorylation of GSK3ß were analyzed by western blotting. ( B ) Primary rat PASMCs transiently transfected with GSK3ß wild type (GSK3ß WT), constitutively active GSK3ß (GSK3ß S9A), empty vector (pcDNA3.1 TOPO) and proliferation was assessed after stimulation with 10% FCS for 24 hrs by [ 3 H]-thymidine incorporation. All values are expressed as mean ± SEM (n = 6, WT or S9A n = 12). Data were expressed as counts per minute (cpm) and normalized to the amount of cells per well. Values were presented significant as ***P

    Article Snippet: MiniPrep Plasmid Isolation Kit was obtained from PeqLab (Erlangen, Germany) and MaxiPrep Plasmid Isolation Kit, PCR purification Kit was purchased from QIAGEN (Hilden, Germany), pGEMT-Easy vector Kit was obtained from Promega, (Mannheim, Germany), pcDNA3.1 Directional TOPO Expression Kit, Lipofectamine 2000, T4 Ligase, Platinum Taq DNA Polymerase High Fidelity, Platinum SYBR Green qPCR SuperMix-UDG was purchased from Invitrogen (Karlsruhe, Germany), restriction enzymes (BamHI, BsmI, EcoRV) was purchased from New England BioLabs (Frankfurt, Germany).

    Techniques: Over Expression, Modification, Transfection, Plasmid Preparation, Expressing, Western Blot

    AID and Ung are critical for the generation of staggered DSBs and CSR. A , Forced expression of Aid results in occurrence of staggered DSBs. Paired samples of low Aid expression 4D11 B cells were transfected with an empty pcDNA3.1 vector or a pcDNA3.1 vector containing human Aid cDNA, and then selected in a 21-day culture in the presence of G418. The selected cells were further stimulated for three days with agonistic anti-huCD40 mAb and huIL-4, and harvested to prepare genomic DNA and RNA. To analyze DSBs, blunt or total (blunt plus staggered) Sμ upstream DSB ends were amplified by LM-PCR after pre-treatment of genomic DNA with nil (T4−) or T4 pol (T4+) before being ligated with BW linker. Linker-ligated genomic DNA (8 ng from 1280 B cells) was serially two-fold diluted into unligated homologous genomic DNA and used as template in LM-PCR (lanes 1–8 in each panel). Germline Iμ-Cμ, Iγ1-Cγ1, circle Iγ1-Cμ, mature V H DJ H -Cμ1, total Aid or expression vector-encoded Aid (vector Aid ), and β- actin transcripts were analyzed by RT-PCR using serially two-fold diluted cDNA as templates. B , Expression of DN Aid abrogates the generation of staggered DNA ends. Paired samples of spontaneously switching 4B6 B cells transfected with an empty pcDNA3.1 vector or a pcDNA3.1 vector containing a human DN Aid construct and then selected in a 21 days culture in the presence of G418. The selected cells were further cultured with nil for three days and then harvested to prepare genomic DNA and RNA. To analyze DSBs, blunt or total (blunt plus staggered) Sμ upstream DSB ends were amplified by LM-PCR after pre-treatment of genomic DNA with nil (T4−) or T4 pol (T4+) before being ligated with BW linker. Linker-ligated genomic DNA (8 ng from 1280 B cells) was serially two-fold diluted into unligated homologous genomic DNA and used as template in LM-PCR (lanes 1–8 in each panel). Germline Iμ-Cμ, Iγ1-Cγ1, circle Iγ1-Cμ, mature V H DJ H -Cγ1, total Aid or expression vector-encoded DN Aid , and β- actin transcripts were analyzed by RT-PCR using serially two-fold diluted cDNA as templates. C , Expression of Ugi abrogates the emergence of staggered DSBs. Paired samples of spontaneously switching human 4B6 B cells or CSR-inducible human 3G10 B cells were transfected with an empty pcDNA3.1 vector or a pcDNA3.1 vector containing an Ugi construct and selected in a 21 days culture in the presence of G418. Genomic DNA, RNA and whole-cell extract were prepared from transfected 4B6 B cells cultured with nil for three days and transfected 3G10 B cells stimulated for three day with nil or agonistic anti-huCD40 mAb and huIL-4. To analyze DSBs, blunt or total (blunt plus staggered) Sμ upstream DSB ends were amplified by LM-PCR after pre-treatment of genomic DNA with nil (T4−) or T4 pol (T4+) before being ligated with BW linker. Linker-ligated genomic DNA (8 ng from 1280 B cells) was serially two-fold diluted into unligated homologous genomic DNA and used as template in LM-PCR (lanes 1–8 in each panel). Ung activity of the B cells was detected by incubate 10 μg, 1.0 μg or 0.1 μg (protein) of clarified whole-cell extract with a [ 32 P] labeled double-stranded oligonucleotide containing a single dU/dG residue. The reaction products were resolved in 15% TBE-urea polyacrylamide gels. Germline Iμ-Cμ, Iγ1-Cγ1, circle Iγ1-Cμ, mature V H DJ H -Cγ1, Aid and β- actin transcripts were analyzed by RT-PCR using serially two-fold diluted cDNA as templates. D , Intra-S region recombination occurs in the absence of AID or Ung. DNA was PCR-amplified from a mouse germline S□ region, as inserted into pCR-Blunt II-TOPO® plasmid (left panel) or genomic DNA isolated from aicda +/+ and aicda −/− (center panel) or ung +/+ and ung −/− (right panel) mouse B cells stimulated by LPS and moIL-4 for three days (left, center and right panels were from different gels), and specified by Southern-blot using a specific [ 32 P]-labeled Sμ probe. Recombined intra-Sμ DNA resulted in Sμ DNA amplification products smaller than those expected based on germline Sμ (3730 bp) region length (closed arrowhead). Migration of molecular weight markers is indicated by open arrowheads.

    Journal: Molecular immunology

    Article Title: AID- and Ung-dependent generation of staggered double-strand DNA breaks in immunoglobulin class switch DNA recombination: a post-cleavage role for AID

    doi: 10.1016/j.molimm.2008.07.003

    Figure Lengend Snippet: AID and Ung are critical for the generation of staggered DSBs and CSR. A , Forced expression of Aid results in occurrence of staggered DSBs. Paired samples of low Aid expression 4D11 B cells were transfected with an empty pcDNA3.1 vector or a pcDNA3.1 vector containing human Aid cDNA, and then selected in a 21-day culture in the presence of G418. The selected cells were further stimulated for three days with agonistic anti-huCD40 mAb and huIL-4, and harvested to prepare genomic DNA and RNA. To analyze DSBs, blunt or total (blunt plus staggered) Sμ upstream DSB ends were amplified by LM-PCR after pre-treatment of genomic DNA with nil (T4−) or T4 pol (T4+) before being ligated with BW linker. Linker-ligated genomic DNA (8 ng from 1280 B cells) was serially two-fold diluted into unligated homologous genomic DNA and used as template in LM-PCR (lanes 1–8 in each panel). Germline Iμ-Cμ, Iγ1-Cγ1, circle Iγ1-Cμ, mature V H DJ H -Cμ1, total Aid or expression vector-encoded Aid (vector Aid ), and β- actin transcripts were analyzed by RT-PCR using serially two-fold diluted cDNA as templates. B , Expression of DN Aid abrogates the generation of staggered DNA ends. Paired samples of spontaneously switching 4B6 B cells transfected with an empty pcDNA3.1 vector or a pcDNA3.1 vector containing a human DN Aid construct and then selected in a 21 days culture in the presence of G418. The selected cells were further cultured with nil for three days and then harvested to prepare genomic DNA and RNA. To analyze DSBs, blunt or total (blunt plus staggered) Sμ upstream DSB ends were amplified by LM-PCR after pre-treatment of genomic DNA with nil (T4−) or T4 pol (T4+) before being ligated with BW linker. Linker-ligated genomic DNA (8 ng from 1280 B cells) was serially two-fold diluted into unligated homologous genomic DNA and used as template in LM-PCR (lanes 1–8 in each panel). Germline Iμ-Cμ, Iγ1-Cγ1, circle Iγ1-Cμ, mature V H DJ H -Cγ1, total Aid or expression vector-encoded DN Aid , and β- actin transcripts were analyzed by RT-PCR using serially two-fold diluted cDNA as templates. C , Expression of Ugi abrogates the emergence of staggered DSBs. Paired samples of spontaneously switching human 4B6 B cells or CSR-inducible human 3G10 B cells were transfected with an empty pcDNA3.1 vector or a pcDNA3.1 vector containing an Ugi construct and selected in a 21 days culture in the presence of G418. Genomic DNA, RNA and whole-cell extract were prepared from transfected 4B6 B cells cultured with nil for three days and transfected 3G10 B cells stimulated for three day with nil or agonistic anti-huCD40 mAb and huIL-4. To analyze DSBs, blunt or total (blunt plus staggered) Sμ upstream DSB ends were amplified by LM-PCR after pre-treatment of genomic DNA with nil (T4−) or T4 pol (T4+) before being ligated with BW linker. Linker-ligated genomic DNA (8 ng from 1280 B cells) was serially two-fold diluted into unligated homologous genomic DNA and used as template in LM-PCR (lanes 1–8 in each panel). Ung activity of the B cells was detected by incubate 10 μg, 1.0 μg or 0.1 μg (protein) of clarified whole-cell extract with a [ 32 P] labeled double-stranded oligonucleotide containing a single dU/dG residue. The reaction products were resolved in 15% TBE-urea polyacrylamide gels. Germline Iμ-Cμ, Iγ1-Cγ1, circle Iγ1-Cμ, mature V H DJ H -Cγ1, Aid and β- actin transcripts were analyzed by RT-PCR using serially two-fold diluted cDNA as templates. D , Intra-S region recombination occurs in the absence of AID or Ung. DNA was PCR-amplified from a mouse germline S□ region, as inserted into pCR-Blunt II-TOPO® plasmid (left panel) or genomic DNA isolated from aicda +/+ and aicda −/− (center panel) or ung +/+ and ung −/− (right panel) mouse B cells stimulated by LPS and moIL-4 for three days (left, center and right panels were from different gels), and specified by Southern-blot using a specific [ 32 P]-labeled Sμ probe. Recombined intra-Sμ DNA resulted in Sμ DNA amplification products smaller than those expected based on germline Sμ (3730 bp) region length (closed arrowhead). Migration of molecular weight markers is indicated by open arrowheads.

    Article Snippet: Aid or dominant negative (DN) Aid transcripts, as encoded by the pcDNA3.1 (pcDNA3.1/V5-His TOPO TA Expression Kit, Invitrogen Corp.)-based expression vector, were amplified using a pcDNA3.1 vector forward primer 5'-ACGACTC ACTATAGGGAGACC-3' specific for a sequence downstream of the transcriptional start site together with the Aid reverse primer huAID-R ( ).

    Techniques: Expressing, Transfection, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Construct, Cell Culture, Activity Assay, Labeling, Isolation, Southern Blot, Migration, Molecular Weight

    Long-term exposure of kisspeptin inhibits insulin secretion in NIT-1 cells. ( a ) NIT-1 cells were transfected with or without pcDNA3.1 + mKiss1-T2A-GFP plasmid, then fixed and stained with Hoechst 33342. Nucleus and GFP signal are shown in blue and green, respectively. The overlay of signals was processed by ImageJ. ( b ) Representative blots of GFP and mouse kisspeptin from transfected NIT-1 cells are shown. ( c ) Insulin secretion ability of control and transfected NIT-1 cells were determined by the amount of secreted luciferase under basal and glucose-stimulated condition. The relative luciferase activity was normalized by total protein in cell lysates. Data represent the means ± standard errors of the mean (n = 3). *Compared with the basal level in the control group; ** p

    Journal: Scientific Reports

    Article Title: Kisspeptin-Activated Autophagy Independently Suppresses Non-Glucose-Stimulated Insulin Secretion from Pancreatic β-Cells

    doi: 10.1038/s41598-019-53826-7

    Figure Lengend Snippet: Long-term exposure of kisspeptin inhibits insulin secretion in NIT-1 cells. ( a ) NIT-1 cells were transfected with or without pcDNA3.1 + mKiss1-T2A-GFP plasmid, then fixed and stained with Hoechst 33342. Nucleus and GFP signal are shown in blue and green, respectively. The overlay of signals was processed by ImageJ. ( b ) Representative blots of GFP and mouse kisspeptin from transfected NIT-1 cells are shown. ( c ) Insulin secretion ability of control and transfected NIT-1 cells were determined by the amount of secreted luciferase under basal and glucose-stimulated condition. The relative luciferase activity was normalized by total protein in cell lysates. Data represent the means ± standard errors of the mean (n = 3). *Compared with the basal level in the control group; ** p

    Article Snippet: The pcDNA3.1(+)-mKiss1-T2A-GFP plasmid was generated first by composing the backbone DNA, the pcDNA3.1(+) mammalian expression vector (Thermo Fisher Scientific), and the insert DNA of mouse Kiss1 -Hiss-tag.

    Techniques: Transfection, Plasmid Preparation, Staining, Luciferase, Activity Assay

    Long-term exposure of kisspeptin decreases (pro)insulin protein level and activates autophagy in NIT-1 cells. Representative blots ( a ) and mRNA levels of insulin ( c ) in NIT-1 cells after transfecting pcDNA3.1 + mKiss1-T2A-GFP for 72 h. Quantifications of blots and mRNA normalized by β-actin or RPL19 are shown as the means ± standard errors of the mean (n = 3). ( b ) Representative blots of autophagy flux marker in cultured NIT-1 cells after overexpressing Kiss1 with/without treating with bafilomycin A1 (20 nM, 3 h). Quantifications of LC3-II normalized by β-actin are shown as the means ± standard errors of the mean (n = 3), which represents autophagy flux. *Compared with level of the control; * p

    Journal: Scientific Reports

    Article Title: Kisspeptin-Activated Autophagy Independently Suppresses Non-Glucose-Stimulated Insulin Secretion from Pancreatic β-Cells

    doi: 10.1038/s41598-019-53826-7

    Figure Lengend Snippet: Long-term exposure of kisspeptin decreases (pro)insulin protein level and activates autophagy in NIT-1 cells. Representative blots ( a ) and mRNA levels of insulin ( c ) in NIT-1 cells after transfecting pcDNA3.1 + mKiss1-T2A-GFP for 72 h. Quantifications of blots and mRNA normalized by β-actin or RPL19 are shown as the means ± standard errors of the mean (n = 3). ( b ) Representative blots of autophagy flux marker in cultured NIT-1 cells after overexpressing Kiss1 with/without treating with bafilomycin A1 (20 nM, 3 h). Quantifications of LC3-II normalized by β-actin are shown as the means ± standard errors of the mean (n = 3), which represents autophagy flux. *Compared with level of the control; * p

    Article Snippet: The pcDNA3.1(+)-mKiss1-T2A-GFP plasmid was generated first by composing the backbone DNA, the pcDNA3.1(+) mammalian expression vector (Thermo Fisher Scientific), and the insert DNA of mouse Kiss1 -Hiss-tag.

    Techniques: Marker, Cell Culture