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  • 99
    New England Biolabs bamhi
    Bamhi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna3 1
    HSPA1L and GR protein levels in decidualized human endometrial stromal fibroblasts. Cultured ESFs were transfected with WT or Ala268Thr HSPA1L <t>-pcDNA3.1</t> constructs or with empty pcDNA3.1 vector (control). Cells were treated with decidualization media supplemented with 100nM dexamethasone (glucocorticoids) for 72h. Both cytosolic and nuclear protein were extracted, and HSPA1L and GR protein levels were measured by Western blot. Band intensity of HSPA1L or GR was normalized to band intensity of the corresponding β-actin. Cytosolic (A) and nuclear (B) HSPA1L levels as well as cytosolic (C) and nuclear (D) GR levels are shown for control (empty vector), WT and Ala268Thr sample groups. Each experiment was performed as triplicates in three different passages (n = 9 each group, except n = 8 for nuclear control group) and bars represent mean + SEM. Significant p-value
    Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 51881 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher trizol reagent
    HSPA1L and GR protein levels in decidualized human endometrial stromal fibroblasts. Cultured ESFs were transfected with WT or Ala268Thr HSPA1L <t>-pcDNA3.1</t> constructs or with empty pcDNA3.1 vector (control). Cells were treated with decidualization media supplemented with 100nM dexamethasone (glucocorticoids) for 72h. Both cytosolic and nuclear protein were extracted, and HSPA1L and GR protein levels were measured by Western blot. Band intensity of HSPA1L or GR was normalized to band intensity of the corresponding β-actin. Cytosolic (A) and nuclear (B) HSPA1L levels as well as cytosolic (C) and nuclear (D) GR levels are shown for control (empty vector), WT and Ala268Thr sample groups. Each experiment was performed as triplicates in three different passages (n = 9 each group, except n = 8 for nuclear control group) and bars represent mean + SEM. Significant p-value
    Trizol Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 635441 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna 3 1
    Effect of Cx43 siRNA and overexpression of pcDNA ™ 3.1- Cx43 on the Cx43 expression in saphenous vein smooth muscle cells. (A) VSMCs were transfected with control-siRNA or empty plasmid or pcDNA ™ 3.1- Cx43 or Cx43-siRNA for 48 h. (B) SV SMCs were transfected with siRNA against Cx43 and non-silencing siRNA and total protein expression of Cx43 was determined by Western blot analysis after 48h and 72h. The cells which were transfected with Cx43-siRNA were treated with Ang II (10 −7  mol/L) for 24 h, lysed and protein expression of Cx43 was measured. Values are mean ± SE for three independent experiments. § P
    Pcdna 3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10361 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bamhi
    Direct visualization of <t>HPV88</t> on an EtBr-stained gel, after digestion with <t>BamHI,</t> lane 1. Lane M, Gene Ruler 1 kb DNA ladder (Fermentas).
    Bamhi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine ltx
    IFA results of PCV1-free PK-15 cells transfected with the ligation mixtures of linear TTSuV2 genomic DNA derived from clone pSC-PTTV2c (A) or pSC-TTV2-#471942 (C) with plasmid pSC-2PTTV2c-RR (B) or pSC-2PTTV2b-RR (D) or with <t>Lipofectamine</t> <t>LTX</t> only (E).
    Lipofectamine Ltx, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 30153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gateway lr clonase ii enzyme mix
    IFA results of PCV1-free PK-15 cells transfected with the ligation mixtures of linear TTSuV2 genomic DNA derived from clone pSC-PTTV2c (A) or pSC-TTV2-#471942 (C) with plasmid pSC-2PTTV2c-RR (B) or pSC-2PTTV2b-RR (D) or with <t>Lipofectamine</t> <t>LTX</t> only (E).
    Gateway Lr Clonase Ii Enzyme Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5539 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 293ft cells
    TBX5 is a functional target of miR-10b ( A ) qPCR of miR-10b, TBX5 , DYRK1A , and PTEN in <t>293FT</t> cells transfected with miR-10b and TBX5, alone or in combination. ( B ) qPCR of miR-10b, TBX5 , DYRK1A , and PTEN in 293FT cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. ( C ) qPCR of miR-10b in LM2 cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. ( D ) Immunoblotting of TBX5, DYRK1A, PTEN, phospho-AKT, AKT, and HSP90 in LM2 cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. ( E, F ) Growth curves ( E ) and migration and invasion assays ( F ) of LM2 cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. n = 4 and 3 wells per group in ( E ) and ( F ), respectively. P values were from a two-tailed, unpaired t -test.
    293ft Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10341 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    OriGene bamhi
    TBX5 is a functional target of miR-10b ( A ) qPCR of miR-10b, TBX5 , DYRK1A , and PTEN in <t>293FT</t> cells transfected with miR-10b and TBX5, alone or in combination. ( B ) qPCR of miR-10b, TBX5 , DYRK1A , and PTEN in 293FT cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. ( C ) qPCR of miR-10b in LM2 cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. ( D ) Immunoblotting of TBX5, DYRK1A, PTEN, phospho-AKT, AKT, and HSP90 in LM2 cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. ( E, F ) Growth curves ( E ) and migration and invasion assays ( F ) of LM2 cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. n = 4 and 3 wells per group in ( E ) and ( F ), respectively. P values were from a two-tailed, unpaired t -test.
    Bamhi, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 700 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sf9 cells
    Binding and blocking of BNeV VLPs to synthetic HBGAs. (A) Ninety-six-well plates were coated with 50 μg/ml BNeV VLPs, GST-tagged-VP8* protein and GST-tagged P particles (10 μg/ml), supernatant of wild-type baculovirus-infected <t>Sf9</t> cells lysate, and GST and then incubated with each of the synthetic HBGAs (10 μg/ml). The binding of each HBGA to target viral proteins and the control was determined by addition of horseradish peroxidase-conjugated streptavidin as described in Materials and Methods. (B) α1,2-Linked fucose, α1,3/4-linked fucose, αGal, and GalNAc epitopes were removed from each synthetic HBGA coated in each well using the corresponding enzyme. After incubation of BNeV VLPs at 50 μg/ml, the binding of BNeV VLPs was determined using hyperimmune serum against BNeV capsid protein, followed by addition of horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. The signal intensities for graphs in panels A and B were visualized using TMB at 450 nm in three independent experiments. Error bars indicate SD from triplicate samples.
    Sf9 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11677 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa bamhi
    Binding and blocking of BNeV VLPs to synthetic HBGAs. (A) Ninety-six-well plates were coated with 50 μg/ml BNeV VLPs, GST-tagged-VP8* protein and GST-tagged P particles (10 μg/ml), supernatant of wild-type baculovirus-infected <t>Sf9</t> cells lysate, and GST and then incubated with each of the synthetic HBGAs (10 μg/ml). The binding of each HBGA to target viral proteins and the control was determined by addition of horseradish peroxidase-conjugated streptavidin as described in Materials and Methods. (B) α1,2-Linked fucose, α1,3/4-linked fucose, αGal, and GalNAc epitopes were removed from each synthetic HBGA coated in each well using the corresponding enzyme. After incubation of BNeV VLPs at 50 μg/ml, the binding of BNeV VLPs was determined using hyperimmune serum against BNeV capsid protein, followed by addition of horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. The signal intensities for graphs in panels A and B were visualized using TMB at 450 nm in three independent experiments. Error bars indicate SD from triplicate samples.
    Bamhi, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 6140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa ptre tight vector
    Binding and blocking of BNeV VLPs to synthetic HBGAs. (A) Ninety-six-well plates were coated with 50 μg/ml BNeV VLPs, GST-tagged-VP8* protein and GST-tagged P particles (10 μg/ml), supernatant of wild-type baculovirus-infected <t>Sf9</t> cells lysate, and GST and then incubated with each of the synthetic HBGAs (10 μg/ml). The binding of each HBGA to target viral proteins and the control was determined by addition of horseradish peroxidase-conjugated streptavidin as described in Materials and Methods. (B) α1,2-Linked fucose, α1,3/4-linked fucose, αGal, and GalNAc epitopes were removed from each synthetic HBGA coated in each well using the corresponding enzyme. After incubation of BNeV VLPs at 50 μg/ml, the binding of BNeV VLPs was determined using hyperimmune serum against BNeV capsid protein, followed by addition of horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. The signal intensities for graphs in panels A and B were visualized using TMB at 450 nm in three independent experiments. Error bars indicate SD from triplicate samples.
    Ptre Tight Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 983 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher streptomycin
    Binding and blocking of BNeV VLPs to synthetic HBGAs. (A) Ninety-six-well plates were coated with 50 μg/ml BNeV VLPs, GST-tagged-VP8* protein and GST-tagged P particles (10 μg/ml), supernatant of wild-type baculovirus-infected <t>Sf9</t> cells lysate, and GST and then incubated with each of the synthetic HBGAs (10 μg/ml). The binding of each HBGA to target viral proteins and the control was determined by addition of horseradish peroxidase-conjugated streptavidin as described in Materials and Methods. (B) α1,2-Linked fucose, α1,3/4-linked fucose, αGal, and GalNAc epitopes were removed from each synthetic HBGA coated in each well using the corresponding enzyme. After incubation of BNeV VLPs at 50 μg/ml, the binding of BNeV VLPs was determined using hyperimmune serum against BNeV capsid protein, followed by addition of horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. The signal intensities for graphs in panels A and B were visualized using TMB at 450 nm in three independent experiments. Error bars indicate SD from triplicate samples.
    Streptomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 108183 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    HSPA1L and GR protein levels in decidualized human endometrial stromal fibroblasts. Cultured ESFs were transfected with WT or Ala268Thr HSPA1L -pcDNA3.1 constructs or with empty pcDNA3.1 vector (control). Cells were treated with decidualization media supplemented with 100nM dexamethasone (glucocorticoids) for 72h. Both cytosolic and nuclear protein were extracted, and HSPA1L and GR protein levels were measured by Western blot. Band intensity of HSPA1L or GR was normalized to band intensity of the corresponding β-actin. Cytosolic (A) and nuclear (B) HSPA1L levels as well as cytosolic (C) and nuclear (D) GR levels are shown for control (empty vector), WT and Ala268Thr sample groups. Each experiment was performed as triplicates in three different passages (n = 9 each group, except n = 8 for nuclear control group) and bars represent mean + SEM. Significant p-value

    Journal: PLoS Genetics

    Article Title: Whole exome sequencing reveals HSPA1L as a genetic risk factor for spontaneous preterm birth

    doi: 10.1371/journal.pgen.1007394

    Figure Lengend Snippet: HSPA1L and GR protein levels in decidualized human endometrial stromal fibroblasts. Cultured ESFs were transfected with WT or Ala268Thr HSPA1L -pcDNA3.1 constructs or with empty pcDNA3.1 vector (control). Cells were treated with decidualization media supplemented with 100nM dexamethasone (glucocorticoids) for 72h. Both cytosolic and nuclear protein were extracted, and HSPA1L and GR protein levels were measured by Western blot. Band intensity of HSPA1L or GR was normalized to band intensity of the corresponding β-actin. Cytosolic (A) and nuclear (B) HSPA1L levels as well as cytosolic (C) and nuclear (D) GR levels are shown for control (empty vector), WT and Ala268Thr sample groups. Each experiment was performed as triplicates in three different passages (n = 9 each group, except n = 8 for nuclear control group) and bars represent mean + SEM. Significant p-value

    Article Snippet: The HSPA1L sequences were subcloned into pcDNA3.1(+) vector (Invitrogen) by ligating into the BamHI/NotI sites using Quick ligase (New England Biolabs).

    Techniques: Cell Culture, Transfection, Construct, Plasmid Preparation, Western Blot

    Generation of prM-E cell line for VLP production. ZIKV prM-E was PCR amplified by use of specific primers, using the codon-optimized C-prM-E construct as the template (A), and cloned into the pCDNA3.1 expression vector (B). (C) E protein expression was determined by fluorescence microscopy after staining with the MAB10216 antibody. (D) Culture supernatants were harvested from ZIKV prM-E-expressing cells and ultracentrifuged. Cell and virus pellets were lysed, and E protein expression was determined by Western blotting. (E) ZIKV prM-E was PCR amplified and cloned into the lentiviral vector pLenti6/5-D-Topo. 293T cells were then transfected with the pLenti-prM-E construct along with the helper plasmid and VSV-G envelope, and ZIKV prM-E lentiviral particles were harvested at 48 h posttransfection. 293T cells were then transduced with the lentiviral particles, and cells were either bulk selected or selected as single-cell clones by culture in the presence of blasticidin. Selected cells were confirmed to have E protein expression via immunofluorescence assay after staining with the MAB10216 antibody (F) and by flow cytometry (G). The mean fluorescence intensity (MFI) of E protein expression for each prM-E clone is indicated on the right. (H) The indicated pLenti-ZIKV-prM-E cell clones were seeded in equal cell numbers, and culture supernatants were harvested and ultracentrifuged. VLP pellets were lysed and resolved by SDS-PAGE, and E protein expression was determined by Western blotting.

    Journal: Journal of Virology

    Article Title: Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

    doi: 10.1128/JVI.00834-17

    Figure Lengend Snippet: Generation of prM-E cell line for VLP production. ZIKV prM-E was PCR amplified by use of specific primers, using the codon-optimized C-prM-E construct as the template (A), and cloned into the pCDNA3.1 expression vector (B). (C) E protein expression was determined by fluorescence microscopy after staining with the MAB10216 antibody. (D) Culture supernatants were harvested from ZIKV prM-E-expressing cells and ultracentrifuged. Cell and virus pellets were lysed, and E protein expression was determined by Western blotting. (E) ZIKV prM-E was PCR amplified and cloned into the lentiviral vector pLenti6/5-D-Topo. 293T cells were then transfected with the pLenti-prM-E construct along with the helper plasmid and VSV-G envelope, and ZIKV prM-E lentiviral particles were harvested at 48 h posttransfection. 293T cells were then transduced with the lentiviral particles, and cells were either bulk selected or selected as single-cell clones by culture in the presence of blasticidin. Selected cells were confirmed to have E protein expression via immunofluorescence assay after staining with the MAB10216 antibody (F) and by flow cytometry (G). The mean fluorescence intensity (MFI) of E protein expression for each prM-E clone is indicated on the right. (H) The indicated pLenti-ZIKV-prM-E cell clones were seeded in equal cell numbers, and culture supernatants were harvested and ultracentrifuged. VLP pellets were lysed and resolved by SDS-PAGE, and E protein expression was determined by Western blotting.

    Article Snippet: The prM-E construct was generated by PCR amplification of the prM-E region spanning amino acids 105 to 795, using a Phusion high-fidelity PCR kit (New England BioLabs), and was cloned into the pcDNA3.1+ vector (Invitrogen).

    Techniques: Polymerase Chain Reaction, Amplification, Construct, Clone Assay, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Staining, Western Blot, Transfection, Transduction, Immunofluorescence, Flow Cytometry, Cytometry, SDS Page

    Expression of ZIKV C-prM-E by use of a codon-optimized synthetic construct. (A) A codon-optimized ZIKV C-prM-E gene was synthesized via gene synthesis technology, using the sequence available from the current outbreak in the Americas, and cloned into the pcDNA3.1 vector. (B) 293T cells were transfected with the ZIKV C-prM-E or WNV C-prM-E construct. Cells were fixed, stained with the MAB10216 and MAB8150 antibodies, and analyzed for E protein expression by fluorescence microscopy. (C) 293T cells were transfected as described above. At 48 h posttransfection, cells were radiolabeled with [ 35 S]Met/Cys. Cell lysates were immunoprecipitated with protein A beads coated with MAB10216 or anti-WNV serum, resolved by SDS-PAGE, and visualized by PhosphorImager analysis.

    Journal: Journal of Virology

    Article Title: Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

    doi: 10.1128/JVI.00834-17

    Figure Lengend Snippet: Expression of ZIKV C-prM-E by use of a codon-optimized synthetic construct. (A) A codon-optimized ZIKV C-prM-E gene was synthesized via gene synthesis technology, using the sequence available from the current outbreak in the Americas, and cloned into the pcDNA3.1 vector. (B) 293T cells were transfected with the ZIKV C-prM-E or WNV C-prM-E construct. Cells were fixed, stained with the MAB10216 and MAB8150 antibodies, and analyzed for E protein expression by fluorescence microscopy. (C) 293T cells were transfected as described above. At 48 h posttransfection, cells were radiolabeled with [ 35 S]Met/Cys. Cell lysates were immunoprecipitated with protein A beads coated with MAB10216 or anti-WNV serum, resolved by SDS-PAGE, and visualized by PhosphorImager analysis.

    Article Snippet: The prM-E construct was generated by PCR amplification of the prM-E region spanning amino acids 105 to 795, using a Phusion high-fidelity PCR kit (New England BioLabs), and was cloned into the pcDNA3.1+ vector (Invitrogen).

    Techniques: Expressing, Construct, Synthesized, Sequencing, Clone Assay, Plasmid Preparation, Transfection, Staining, Fluorescence, Microscopy, Immunoprecipitation, SDS Page

    Expression of ZIKV prM-E alone releases VLPs into the supernatant, while C-prM-E requires the NS2B-3 protease for efficient VLP release. (A) 293T cells were transfected with the pCDNA3.1 vector expressing ZIKV prM-E or C-prM-E along with the indicated expression vectors. Cells were radiolabeled with [ 35 S]Met/Cys, and culture supernatants were harvested and ultracentrifuged. Cell and virion samples were lysed and immunoprecipitated with MAB10216-coated protein A beads, resolved by SDS-PAGE, and visualized by PhosphorImager analysis. (B) Culture supernatants were harvested from cells expressing ZIKV C-prM-E or prM-E as indicated in Materials and Methods. Twenty-five to 30 ml of supernatant was transferred to ultracentrifuge tubes and carefully underlaid with 5 ml of 25% glycerol in TNE buffer. VLPs were pelleted by centrifugation at 110,500 × g for 3 h at 4°C. Thereafter, the supernatant was carefully removed and the VLP pellet resuspended in TNE buffer. An aliquot of concentrated VLPs was subjected to lysis with 10× RIPA buffer, and E protein in the preparations was detected by Western blotting. (C) VLPs were concentrated as described above, and images were acquired after negative staining, using a JEOL1010 transmission electron microscope with a Hamamatsu digital camera. Bars, ∼30 nm. (D and E) Immunization studies in mice. (D) BALB/c mice were divided into groups of six mice each. Mice received the primary immunization on day 0, followed by 2 boosters, at days 14 and 28, and were finally sacrificed at day 63 post-primary immunization. (E) Mice were divided into 5 groups and received immunizations with either C-prM-E/prM-E DNA or VLPs. For DNA immunization, a total of 50 μg of DNA in a volume of 100 μl PBS was injected intramuscularly (i.m.). For VLPs, the first immunization consisted of a VLP preparation mixed with TiterMax Gold adjuvant in a total volume of 100 μl injected intramuscularly. For subsequent boosters, mice received VLPs alone, without adjuvant. Control mice were injected with PBS.

    Journal: Journal of Virology

    Article Title: Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

    doi: 10.1128/JVI.00834-17

    Figure Lengend Snippet: Expression of ZIKV prM-E alone releases VLPs into the supernatant, while C-prM-E requires the NS2B-3 protease for efficient VLP release. (A) 293T cells were transfected with the pCDNA3.1 vector expressing ZIKV prM-E or C-prM-E along with the indicated expression vectors. Cells were radiolabeled with [ 35 S]Met/Cys, and culture supernatants were harvested and ultracentrifuged. Cell and virion samples were lysed and immunoprecipitated with MAB10216-coated protein A beads, resolved by SDS-PAGE, and visualized by PhosphorImager analysis. (B) Culture supernatants were harvested from cells expressing ZIKV C-prM-E or prM-E as indicated in Materials and Methods. Twenty-five to 30 ml of supernatant was transferred to ultracentrifuge tubes and carefully underlaid with 5 ml of 25% glycerol in TNE buffer. VLPs were pelleted by centrifugation at 110,500 × g for 3 h at 4°C. Thereafter, the supernatant was carefully removed and the VLP pellet resuspended in TNE buffer. An aliquot of concentrated VLPs was subjected to lysis with 10× RIPA buffer, and E protein in the preparations was detected by Western blotting. (C) VLPs were concentrated as described above, and images were acquired after negative staining, using a JEOL1010 transmission electron microscope with a Hamamatsu digital camera. Bars, ∼30 nm. (D and E) Immunization studies in mice. (D) BALB/c mice were divided into groups of six mice each. Mice received the primary immunization on day 0, followed by 2 boosters, at days 14 and 28, and were finally sacrificed at day 63 post-primary immunization. (E) Mice were divided into 5 groups and received immunizations with either C-prM-E/prM-E DNA or VLPs. For DNA immunization, a total of 50 μg of DNA in a volume of 100 μl PBS was injected intramuscularly (i.m.). For VLPs, the first immunization consisted of a VLP preparation mixed with TiterMax Gold adjuvant in a total volume of 100 μl injected intramuscularly. For subsequent boosters, mice received VLPs alone, without adjuvant. Control mice were injected with PBS.

    Article Snippet: The prM-E construct was generated by PCR amplification of the prM-E region spanning amino acids 105 to 795, using a Phusion high-fidelity PCR kit (New England BioLabs), and was cloned into the pcDNA3.1+ vector (Invitrogen).

    Techniques: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Centrifugation, Lysis, Western Blot, Negative Staining, Transmission Assay, Microscopy, Mouse Assay, Injection

    KIAA0101 tv2 overexpression suppresses the expression level of KIAA0101 tv1 in HCC cells HepG2 and HepG2.2.15 cells were transfected with KIAA0101 tv2 plasmid. shKIAA0101 tv1 was used as the positive control. pcDNA3.1(-) or scrambled shRNA (sh(-)) was used as the negative control. Mock transfection was used as the blank control. The mRNA and protein levels of KIAA0101 tv1 were determined in the indicated cells using (A) real-time RT-PCR and (B) western blot respectively. (C) Luciferase activity assay. KIAA0101 tv2 expression plasmids were transiently co-transfected with either pGL3-KIAA0101 promoter or pGL3-basic into HepG2 cells. Luciferase activity was measured following 48 h of incubation. All graphs show the mean±SD of at least three independent experiments. * P

    Journal: Oncotarget

    Article Title: Variant 2 of KIAA0101, antagonizing its oncogenic variant 1, might be a potential therapeutic strategy in hepatocellular carcinoma

    doi: 10.18632/oncotarget.16702

    Figure Lengend Snippet: KIAA0101 tv2 overexpression suppresses the expression level of KIAA0101 tv1 in HCC cells HepG2 and HepG2.2.15 cells were transfected with KIAA0101 tv2 plasmid. shKIAA0101 tv1 was used as the positive control. pcDNA3.1(-) or scrambled shRNA (sh(-)) was used as the negative control. Mock transfection was used as the blank control. The mRNA and protein levels of KIAA0101 tv1 were determined in the indicated cells using (A) real-time RT-PCR and (B) western blot respectively. (C) Luciferase activity assay. KIAA0101 tv2 expression plasmids were transiently co-transfected with either pGL3-KIAA0101 promoter or pGL3-basic into HepG2 cells. Luciferase activity was measured following 48 h of incubation. All graphs show the mean±SD of at least three independent experiments. * P

    Article Snippet: Plasmids construction The KIAA0101 tv2 coding regions was cloned into the BamH I and Hind III sites of vector pcDNA3.1(-) (Invitrogen, Carlsbad, CA, USA) and pCMV-Tag 2B (Invitrogen) to generate pcDNA3.1(-)-KIAA0101 tv2 and Flag-KIAA0101 tv2 respectively using the primers P1-Forword and P1-Reverse ( ).

    Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Positive Control, shRNA, Negative Control, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Incubation

    KIAA0101 tv2 competes with KIAA0101 tv1 for binding to P53 in mammalian cells (A) 48 hours after transfection, whole cell lysate was immunoprecipitated (IP) using anti-FLAG antibody and subsequently immunoblotted (IB) with anti-P53 antibody. The cell lysate was also immunoprecipitated using anti-P53 antibody and IB with the anti-FLAG antibody. IP lysate (5%) was used as input. (B) Increasing concentrations of KIAA0101 tv2 prevented the co-IP of KIAA0101 tv1 with P53. HEK 293T cells were co-transfected with pcDNA3.1(-)-KIAA0101 tv1 (1 μg), pcDNA3.1(-)-P53 (1 μg), and increasing amounts (0_4 μg) of Flag-KIAA0101 tv2 plasmids. Cell lysate was immunoprecipitated using the anti-FLAG antibody and subsequently IB with anti-P53 and anti-KIAA0101 tv1 antibodies. Cell lysate was also immunoprecipitated using anti-P53 antibody and then IB with antibodies against P53, Flag, and KIAA0101 tv1. Densitometric analysis presented as mean ± SEM. IP lysate (5%) was used as input.

    Journal: Oncotarget

    Article Title: Variant 2 of KIAA0101, antagonizing its oncogenic variant 1, might be a potential therapeutic strategy in hepatocellular carcinoma

    doi: 10.18632/oncotarget.16702

    Figure Lengend Snippet: KIAA0101 tv2 competes with KIAA0101 tv1 for binding to P53 in mammalian cells (A) 48 hours after transfection, whole cell lysate was immunoprecipitated (IP) using anti-FLAG antibody and subsequently immunoblotted (IB) with anti-P53 antibody. The cell lysate was also immunoprecipitated using anti-P53 antibody and IB with the anti-FLAG antibody. IP lysate (5%) was used as input. (B) Increasing concentrations of KIAA0101 tv2 prevented the co-IP of KIAA0101 tv1 with P53. HEK 293T cells were co-transfected with pcDNA3.1(-)-KIAA0101 tv1 (1 μg), pcDNA3.1(-)-P53 (1 μg), and increasing amounts (0_4 μg) of Flag-KIAA0101 tv2 plasmids. Cell lysate was immunoprecipitated using the anti-FLAG antibody and subsequently IB with anti-P53 and anti-KIAA0101 tv1 antibodies. Cell lysate was also immunoprecipitated using anti-P53 antibody and then IB with antibodies against P53, Flag, and KIAA0101 tv1. Densitometric analysis presented as mean ± SEM. IP lysate (5%) was used as input.

    Article Snippet: Plasmids construction The KIAA0101 tv2 coding regions was cloned into the BamH I and Hind III sites of vector pcDNA3.1(-) (Invitrogen, Carlsbad, CA, USA) and pCMV-Tag 2B (Invitrogen) to generate pcDNA3.1(-)-KIAA0101 tv2 and Flag-KIAA0101 tv2 respectively using the primers P1-Forword and P1-Reverse ( ).

    Techniques: Binding Assay, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

    Exogenous expression of KIAA0101 tv2 increases p53 activity (A) HepG2 cells were transiently transfected with the empty plasmid or with the expression plasmid for KIAA0101 tv2. 48 hours after transfection, total mRNA was prepared and subjected to real-time RT-PCR using specific primers. β-actin was used as the control. The experiments were performed at least three times. (B) Western blot. The protein levels of P53, P53 (K382), P53 (S46), and two downstream target genes were assessed in pcDNA3.1(-) or KIAA0101 tv2 expression plasmid-transfected HepG2 cells. All bars show the intensity of the bands quantitated by densitometry based on three independent experiments. * P

    Journal: Oncotarget

    Article Title: Variant 2 of KIAA0101, antagonizing its oncogenic variant 1, might be a potential therapeutic strategy in hepatocellular carcinoma

    doi: 10.18632/oncotarget.16702

    Figure Lengend Snippet: Exogenous expression of KIAA0101 tv2 increases p53 activity (A) HepG2 cells were transiently transfected with the empty plasmid or with the expression plasmid for KIAA0101 tv2. 48 hours after transfection, total mRNA was prepared and subjected to real-time RT-PCR using specific primers. β-actin was used as the control. The experiments were performed at least three times. (B) Western blot. The protein levels of P53, P53 (K382), P53 (S46), and two downstream target genes were assessed in pcDNA3.1(-) or KIAA0101 tv2 expression plasmid-transfected HepG2 cells. All bars show the intensity of the bands quantitated by densitometry based on three independent experiments. * P

    Article Snippet: Plasmids construction The KIAA0101 tv2 coding regions was cloned into the BamH I and Hind III sites of vector pcDNA3.1(-) (Invitrogen, Carlsbad, CA, USA) and pCMV-Tag 2B (Invitrogen) to generate pcDNA3.1(-)-KIAA0101 tv2 and Flag-KIAA0101 tv2 respectively using the primers P1-Forword and P1-Reverse ( ).

    Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot

    Cell growth and adhesion in GKLF transfected EC9706 cells. The cells were transiently transfected with antisense GKLF expression plasmid (AS) and pCDNA3.1 as control (C). The GKLF expression levels detected by RT-PCR were shown in the upper panel. The cell growth rates were shown in the middle panel; each data point represents mean ± S.E.M. of 7 repeats; there was a significant difference between AS and C ( P

    Journal: World Journal of Gastroenterology

    Article Title: Down-regulation of gut-enriched Krüppel-like factor expression in esophageal cancer

    doi: 10.3748/wjg.v8.i6.966

    Figure Lengend Snippet: Cell growth and adhesion in GKLF transfected EC9706 cells. The cells were transiently transfected with antisense GKLF expression plasmid (AS) and pCDNA3.1 as control (C). The GKLF expression levels detected by RT-PCR were shown in the upper panel. The cell growth rates were shown in the middle panel; each data point represents mean ± S.E.M. of 7 repeats; there was a significant difference between AS and C ( P

    Article Snippet: DNA fragment containing GKLF cDNA (-67 to + 1444 nucleotides) was cut out from pMD18T vector by BamH I and Hind III digestion and subcloned into pCDNA3.1 expression vector (Invitrogen, San Diego, CA) in antisense orientation.

    Techniques: Transfection, Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

    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

    Overexpression MKL1 promoted cell migration and invasion in HeLa cells. a HeLa cells were transfected with pMKL1 or pCDNA3.1. The MKL1 expression level was determined by western blot at 24 h and 48 h after transfection. pCDNA3.1 serves as the negative control and GAPDH serves as the loading control. b The effect of MKL1 overexpression on cell migration was determined by wound healing assay. c Quantification of the wound healing assay. d The effect of MKL1 overexpression on cell invasion was determined in a Boyden chamber assay. e The number of cells on the underside of the filter was determined and significantly ( P

    Journal: Cell Communication and Signaling : CCS

    Article Title: LncRNA HOTAIR promotes cell migration and invasion by regulating MKL1 via inhibition miR206 expression in HeLa cells

    doi: 10.1186/s12964-018-0216-3

    Figure Lengend Snippet: Overexpression MKL1 promoted cell migration and invasion in HeLa cells. a HeLa cells were transfected with pMKL1 or pCDNA3.1. The MKL1 expression level was determined by western blot at 24 h and 48 h after transfection. pCDNA3.1 serves as the negative control and GAPDH serves as the loading control. b The effect of MKL1 overexpression on cell migration was determined by wound healing assay. c Quantification of the wound healing assay. d The effect of MKL1 overexpression on cell invasion was determined in a Boyden chamber assay. e The number of cells on the underside of the filter was determined and significantly ( P

    Article Snippet: The fragment was cloned into the mammalian expression vector pCDNA3.1/myc-his B (Invitrogen, USA).

    Techniques: Over Expression, Migration, Transfection, Expressing, Western Blot, Negative Control, Wound Healing Assay, Boyden Chamber Assay

    Effect of Cx43 siRNA and overexpression of pcDNA ™ 3.1- Cx43 on the Cx43 expression in saphenous vein smooth muscle cells. (A) VSMCs were transfected with control-siRNA or empty plasmid or pcDNA ™ 3.1- Cx43 or Cx43-siRNA for 48 h. (B) SV SMCs were transfected with siRNA against Cx43 and non-silencing siRNA and total protein expression of Cx43 was determined by Western blot analysis after 48h and 72h. The cells which were transfected with Cx43-siRNA were treated with Ang II (10 −7  mol/L) for 24 h, lysed and protein expression of Cx43 was measured. Values are mean ± SE for three independent experiments. § P

    Journal: Journal of molecular and cellular cardiology

    Article Title: Involvement of Connexin 43 in Angiotensin II-induced Migration and Proliferation of Saphenous Vein Smooth Muscle Cells via the MAPK-AP-1 Signaling Pathway

    doi: 10.1016/j.yjmcc.2008.03.002

    Figure Lengend Snippet: Effect of Cx43 siRNA and overexpression of pcDNA ™ 3.1- Cx43 on the Cx43 expression in saphenous vein smooth muscle cells. (A) VSMCs were transfected with control-siRNA or empty plasmid or pcDNA ™ 3.1- Cx43 or Cx43-siRNA for 48 h. (B) SV SMCs were transfected with siRNA against Cx43 and non-silencing siRNA and total protein expression of Cx43 was determined by Western blot analysis after 48h and 72h. The cells which were transfected with Cx43-siRNA were treated with Ang II (10 −7 mol/L) for 24 h, lysed and protein expression of Cx43 was measured. Values are mean ± SE for three independent experiments. § P

    Article Snippet: After successful amplification of cDNA from human SV SMCs, the Cx43 product (1149bp) was cloned into pcDNA™ 3.1(+) (5.4kb) (Invitrogen, OR, USA) using BamHI and NotI restriction enzyme sites according to the manufacture’s instruction.

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

    Effect of Cx43 overexpression on the proliferation of VSMCs. After transfection with pcDNA ™ 3.1- Cx43 and empty vector, VSMCs were treated with Ang II (10 −7  mol/L) for 24 h. (A) BrdU incorporation into VSMC was increased by Cx43 overexpression or Ang II stimulation. (B) Cell number of VSMC was significantly increased by Cx43 overexpression or Ang II stimulation. Values are mean± SE for three independent experiments. § P

    Journal: Journal of molecular and cellular cardiology

    Article Title: Involvement of Connexin 43 in Angiotensin II-induced Migration and Proliferation of Saphenous Vein Smooth Muscle Cells via the MAPK-AP-1 Signaling Pathway

    doi: 10.1016/j.yjmcc.2008.03.002

    Figure Lengend Snippet: Effect of Cx43 overexpression on the proliferation of VSMCs. After transfection with pcDNA ™ 3.1- Cx43 and empty vector, VSMCs were treated with Ang II (10 −7 mol/L) for 24 h. (A) BrdU incorporation into VSMC was increased by Cx43 overexpression or Ang II stimulation. (B) Cell number of VSMC was significantly increased by Cx43 overexpression or Ang II stimulation. Values are mean± SE for three independent experiments. § P

    Article Snippet: After successful amplification of cDNA from human SV SMCs, the Cx43 product (1149bp) was cloned into pcDNA™ 3.1(+) (5.4kb) (Invitrogen, OR, USA) using BamHI and NotI restriction enzyme sites according to the manufacture’s instruction.

    Techniques: Over Expression, Transfection, Plasmid Preparation, BrdU Incorporation Assay

    Truncated Protein Expression from Mutant NMD-Irrelevant MARCKS mRNA (A) Schematic diagram of construct Q (MARCKS wild-type genomic DNA) and construct R (MARCKS(−2) deleted genomic DNA). (B) Western blotting (WB) analysis of total protein (30 μg) isolated from transiently transfected cell lines with constructs Q or R. An antibody against FLAG was used as the primary antibody. (C) Western blotting (WB) analysis of total protein (30 μg) isolated from transiently transfected cell lines with the constructs Q or R was performed using an antibody against MARCKS. Enhanced green fluorescent protein (EGFP) was used as a control for transfection efficiency. Comparable results were obtained in at least two independent experiments. Asterisks (*) denote endogenous MARCKS reacted with anti-MARCKS antibody; Con. denotes the control vector pcDNA3.1(+). The dagger indicates uncharacterized protein that did not interfere with experimental interpretations.

    Journal: PLoS Biology

    Article Title: Selective Translational Repression of Truncated Proteins from Frameshift Mutation-Derived mRNAs in Tumors

    doi: 10.1371/journal.pbio.0050109

    Figure Lengend Snippet: Truncated Protein Expression from Mutant NMD-Irrelevant MARCKS mRNA (A) Schematic diagram of construct Q (MARCKS wild-type genomic DNA) and construct R (MARCKS(−2) deleted genomic DNA). (B) Western blotting (WB) analysis of total protein (30 μg) isolated from transiently transfected cell lines with constructs Q or R. An antibody against FLAG was used as the primary antibody. (C) Western blotting (WB) analysis of total protein (30 μg) isolated from transiently transfected cell lines with the constructs Q or R was performed using an antibody against MARCKS. Enhanced green fluorescent protein (EGFP) was used as a control for transfection efficiency. Comparable results were obtained in at least two independent experiments. Asterisks (*) denote endogenous MARCKS reacted with anti-MARCKS antibody; Con. denotes the control vector pcDNA3.1(+). The dagger indicates uncharacterized protein that did not interfere with experimental interpretations.

    Article Snippet: For the expression study of MARCKS, the expression vector pcDNA3.1(+) (Invitrogen) was cut by Hind III, and the FLAG oligonucleotide was inserted at the Hind III site to allow for specific immunodetection, thereby creating the pcDNA-FLAG vector.

    Techniques: Expressing, Mutagenesis, Construct, Western Blot, Isolation, Transfection, Plasmid Preparation

    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

    Immunofluorescence studies in HEK293T cells overexpressing c-myc tagged MasR using MasR antibodies. Images of fluorescence signal corresponding to secondary cyanine (Cy3)-conjugated antibody (A), nuclear Hoechst 33258 staining (B), merged images (C) and bright field (D) are shown. In cells transfected with the pcDNA 3.1/c-myc-MasR construct, antibodies NLS-1531 and sc-54682 generated a similar staining pattern to that generated with the anti c-myc antibody. No staining was observed with any of these antibodies in cells transfected with the empty vector pcDNA 3.1. Antibody sc-135063 was capable of staining the plasma membrane of cells overexpressing the MasR but also generated widespread signals in cells transfected with the empty vector. For antibody AAR-013 there was no staining associated with the cell membrane. Intense immunocytochemical staining of identical distribution and intensity was revealed in cells transfected with the empty vector and those transfected with the c-myc-MasR construct. Images are representative of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies

    doi: 10.1371/journal.pone.0183278

    Figure Lengend Snippet: Immunofluorescence studies in HEK293T cells overexpressing c-myc tagged MasR using MasR antibodies. Images of fluorescence signal corresponding to secondary cyanine (Cy3)-conjugated antibody (A), nuclear Hoechst 33258 staining (B), merged images (C) and bright field (D) are shown. In cells transfected with the pcDNA 3.1/c-myc-MasR construct, antibodies NLS-1531 and sc-54682 generated a similar staining pattern to that generated with the anti c-myc antibody. No staining was observed with any of these antibodies in cells transfected with the empty vector pcDNA 3.1. Antibody sc-135063 was capable of staining the plasma membrane of cells overexpressing the MasR but also generated widespread signals in cells transfected with the empty vector. For antibody AAR-013 there was no staining associated with the cell membrane. Intense immunocytochemical staining of identical distribution and intensity was revealed in cells transfected with the empty vector and those transfected with the c-myc-MasR construct. Images are representative of 3 independent experiments.

    Article Snippet: The construct was synthesized and subcloned into the pcDNA 3.1 (+) mammalian expression vector between BamHI and EcoRI restriction sites by Life Technologies (Grand Island, NY) (pcDNA 3.1/c-myc-MasR).

    Techniques: Immunofluorescence, Fluorescence, Staining, Transfection, Construct, Generated, Plasmid Preparation

    Western blotting of HEK293T cells overexpressing c-myc tagged MasR using anti c-myc tag and three MasR antibodies. Cells were transfected with varying amounts of a plasmid encoding the c-myc-tagged MasR (0.2 and 0.5 μg of pcDNA 3.1/c-myc-MasR).The anti-c-myc antibody detected a band of 37 kDa whose intensity increased as higher amount of DNA was transfected (arrow). Additionally, a ladder of higher molecular weight (MW) immunoreactive bands could be observed (asterisk). sc-54682 antibody reproduced the band pattern obtained with the c-myc antibody (arrow and asterisk). sc-135063 antibody revealed multiple bands close to 37 kDa with one of them of increasing intensity as higher amount of DNA was transfected (arrow) but did not produce a ladder of higher MW immunoreactive bands. High unspecific staining was attained with sc-135063 antibody. AAR-013 antibody revealed a pattern of immunoreactivity not comparable to those obtained with the other antibodies. Detection of β-tubulin was used as a protein loading control. The membranes are representative of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies

    doi: 10.1371/journal.pone.0183278

    Figure Lengend Snippet: Western blotting of HEK293T cells overexpressing c-myc tagged MasR using anti c-myc tag and three MasR antibodies. Cells were transfected with varying amounts of a plasmid encoding the c-myc-tagged MasR (0.2 and 0.5 μg of pcDNA 3.1/c-myc-MasR).The anti-c-myc antibody detected a band of 37 kDa whose intensity increased as higher amount of DNA was transfected (arrow). Additionally, a ladder of higher molecular weight (MW) immunoreactive bands could be observed (asterisk). sc-54682 antibody reproduced the band pattern obtained with the c-myc antibody (arrow and asterisk). sc-135063 antibody revealed multiple bands close to 37 kDa with one of them of increasing intensity as higher amount of DNA was transfected (arrow) but did not produce a ladder of higher MW immunoreactive bands. High unspecific staining was attained with sc-135063 antibody. AAR-013 antibody revealed a pattern of immunoreactivity not comparable to those obtained with the other antibodies. Detection of β-tubulin was used as a protein loading control. The membranes are representative of 3 independent experiments.

    Article Snippet: The construct was synthesized and subcloned into the pcDNA 3.1 (+) mammalian expression vector between BamHI and EcoRI restriction sites by Life Technologies (Grand Island, NY) (pcDNA 3.1/c-myc-MasR).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Molecular Weight, Staining

    Immunofluorescence studies in HEK293T cells overexpressing c-myc tagged MasR using anti c-myc tag antibody. Images of fluorescence signal corresponding to secondary cyanine (Cy3)-conjugated antibody (A), nuclear Hoechst 33258 staining (B), merged images (C) and bright field (D) are shown. The intense immunostaining obtained with the anti c-myc tag antibody indicated strong expression of MasR in HEK293T cells after transfection with the pcDNA 3.1/c-myc-MasR construct. A significant amount of the total MasR pool was present at the plasma membrane. No c-myc staining was observed in cells transfected with the empty vector pcDNA 3.1. Images are representative of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies

    doi: 10.1371/journal.pone.0183278

    Figure Lengend Snippet: Immunofluorescence studies in HEK293T cells overexpressing c-myc tagged MasR using anti c-myc tag antibody. Images of fluorescence signal corresponding to secondary cyanine (Cy3)-conjugated antibody (A), nuclear Hoechst 33258 staining (B), merged images (C) and bright field (D) are shown. The intense immunostaining obtained with the anti c-myc tag antibody indicated strong expression of MasR in HEK293T cells after transfection with the pcDNA 3.1/c-myc-MasR construct. A significant amount of the total MasR pool was present at the plasma membrane. No c-myc staining was observed in cells transfected with the empty vector pcDNA 3.1. Images are representative of 3 independent experiments.

    Article Snippet: The construct was synthesized and subcloned into the pcDNA 3.1 (+) mammalian expression vector between BamHI and EcoRI restriction sites by Life Technologies (Grand Island, NY) (pcDNA 3.1/c-myc-MasR).

    Techniques: Immunofluorescence, Fluorescence, Staining, Immunostaining, Expressing, Transfection, Construct, Plasmid Preparation

    Autophagic clearance of ARpolyQ. NSC34 cells cotransfected with AR.Q46 and pCDNA3 or HSPB8, and treated with vehicle (ethanol, EtOH) or 10 nM T, for 48 h. (A) Immunofluorescence microscopy (IF) analysis (63x magnification) of AR (green); nuclei were stained with DAPI (blue); scale bar: 20 μm. (B) WB and FRA show AR.Q46 total levels and AR.Q46 insoluble fraction respectively. TUBA was used as loading control. Bar graph represents the FRA mean relative optical density computed over 3 independent biological samples for each condition (n = 3) ± SD (* = P

    Journal: Autophagy

    Article Title: Inhibition of retrograde transport modulates misfolded protein accumulation and clearance in motoneuron diseases

    doi: 10.1080/15548627.2017.1308985

    Figure Lengend Snippet: Autophagic clearance of ARpolyQ. NSC34 cells cotransfected with AR.Q46 and pCDNA3 or HSPB8, and treated with vehicle (ethanol, EtOH) or 10 nM T, for 48 h. (A) Immunofluorescence microscopy (IF) analysis (63x magnification) of AR (green); nuclei were stained with DAPI (blue); scale bar: 20 μm. (B) WB and FRA show AR.Q46 total levels and AR.Q46 insoluble fraction respectively. TUBA was used as loading control. Bar graph represents the FRA mean relative optical density computed over 3 independent biological samples for each condition (n = 3) ± SD (* = P

    Article Snippet: The pcDNA3 (Life Technologies, V790-20) plasmid was used to normalize for transfected plasmid DNA amount.

    Techniques: Immunofluorescence, Microscopy, Staining, Western Blot

    Direct visualization of HPV88 on an EtBr-stained gel, after digestion with BamHI, lane 1. Lane M, Gene Ruler 1 kb DNA ladder (Fermentas).

    Journal: International journal of cancer. Journal international du cancer

    Article Title: Cutaneous human papillomavirus 88: Remarkable differences in viral load

    doi: 10.1002/ijc.23115

    Figure Lengend Snippet: Direct visualization of HPV88 on an EtBr-stained gel, after digestion with BamHI, lane 1. Lane M, Gene Ruler 1 kb DNA ladder (Fermentas).

    Article Snippet: Fifty microliters of unamplified extracted DNA from the original tumor of the index patient, containing ~5 × 1010 copies of HPV88, was digested with 20 U of BamHI (Fermentas).

    Techniques: Staining

    IFA results of PCV1-free PK-15 cells transfected with the ligation mixtures of linear TTSuV2 genomic DNA derived from clone pSC-PTTV2c (A) or pSC-TTV2-#471942 (C) with plasmid pSC-2PTTV2c-RR (B) or pSC-2PTTV2b-RR (D) or with Lipofectamine LTX only (E).

    Journal: Journal of Virology

    Article Title: Rescue of a Porcine Anellovirus (Torque Teno Sus Virus 2) from Cloned Genomic DNA in Pigs

    doi: 10.1128/JVI.00175-12

    Figure Lengend Snippet: IFA results of PCV1-free PK-15 cells transfected with the ligation mixtures of linear TTSuV2 genomic DNA derived from clone pSC-PTTV2c (A) or pSC-TTV2-#471942 (C) with plasmid pSC-2PTTV2c-RR (B) or pSC-2PTTV2b-RR (D) or with Lipofectamine LTX only (E).

    Article Snippet: Two-microgram samples of tandem-dimerized clones pSC-2PTTV2b-RR and pSC-2PTTV2c-RR and vectors pTriEx1.1-Neo and pSC-B-amp/kan were directly transfected into the cells, respectively, using Lipofectamine LTX (Invitrogen) according to the manufacturer's protocol.

    Techniques: Immunofluorescence, Transfection, Ligation, Derivative Assay, Plasmid Preparation

    TBX5 is a functional target of miR-10b ( A ) qPCR of miR-10b, TBX5 , DYRK1A , and PTEN in 293FT cells transfected with miR-10b and TBX5, alone or in combination. ( B ) qPCR of miR-10b, TBX5 , DYRK1A , and PTEN in 293FT cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. ( C ) qPCR of miR-10b in LM2 cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. ( D ) Immunoblotting of TBX5, DYRK1A, PTEN, phospho-AKT, AKT, and HSP90 in LM2 cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. ( E, F ) Growth curves ( E ) and migration and invasion assays ( F ) of LM2 cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. n = 4 and 3 wells per group in ( E ) and ( F ), respectively. P values were from a two-tailed, unpaired t -test.

    Journal: Cancer research

    Article Title: Ablation of miR-10b Suppresses Oncogene-Induced Mammary Tumorigenesis and Metastasis and Reactivates Tumor-Suppressive Pathways

    doi: 10.1158/0008-5472.CAN-16-1571

    Figure Lengend Snippet: TBX5 is a functional target of miR-10b ( A ) qPCR of miR-10b, TBX5 , DYRK1A , and PTEN in 293FT cells transfected with miR-10b and TBX5, alone or in combination. ( B ) qPCR of miR-10b, TBX5 , DYRK1A , and PTEN in 293FT cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. ( C ) qPCR of miR-10b in LM2 cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. ( D ) Immunoblotting of TBX5, DYRK1A, PTEN, phospho-AKT, AKT, and HSP90 in LM2 cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. ( E, F ) Growth curves ( E ) and migration and invasion assays ( F ) of LM2 cells transfected with miR-10b antisense inhibitors and TBX5 siRNA, alone or in combination. n = 4 and 3 wells per group in ( E ) and ( F ), respectively. P values were from a two-tailed, unpaired t -test.

    Article Snippet: 293FT cells were from ThermoFisher Scientific in 2012 and the LM2 subline of MDA-MB-231 cells was from Xiang Zhang (Baylor College of Medicine) in 2014.

    Techniques: Functional Assay, Real-time Polymerase Chain Reaction, Transfection, Migration, Two Tailed Test

    Binding and blocking of BNeV VLPs to synthetic HBGAs. (A) Ninety-six-well plates were coated with 50 μg/ml BNeV VLPs, GST-tagged-VP8* protein and GST-tagged P particles (10 μg/ml), supernatant of wild-type baculovirus-infected Sf9 cells lysate, and GST and then incubated with each of the synthetic HBGAs (10 μg/ml). The binding of each HBGA to target viral proteins and the control was determined by addition of horseradish peroxidase-conjugated streptavidin as described in Materials and Methods. (B) α1,2-Linked fucose, α1,3/4-linked fucose, αGal, and GalNAc epitopes were removed from each synthetic HBGA coated in each well using the corresponding enzyme. After incubation of BNeV VLPs at 50 μg/ml, the binding of BNeV VLPs was determined using hyperimmune serum against BNeV capsid protein, followed by addition of horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. The signal intensities for graphs in panels A and B were visualized using TMB at 450 nm in three independent experiments. Error bars indicate SD from triplicate samples.

    Journal: Journal of Virology

    Article Title: Bovine Nebovirus Interacts with a Wide Spectrum of Histo-Blood Group Antigens

    doi: 10.1128/JVI.02160-17

    Figure Lengend Snippet: Binding and blocking of BNeV VLPs to synthetic HBGAs. (A) Ninety-six-well plates were coated with 50 μg/ml BNeV VLPs, GST-tagged-VP8* protein and GST-tagged P particles (10 μg/ml), supernatant of wild-type baculovirus-infected Sf9 cells lysate, and GST and then incubated with each of the synthetic HBGAs (10 μg/ml). The binding of each HBGA to target viral proteins and the control was determined by addition of horseradish peroxidase-conjugated streptavidin as described in Materials and Methods. (B) α1,2-Linked fucose, α1,3/4-linked fucose, αGal, and GalNAc epitopes were removed from each synthetic HBGA coated in each well using the corresponding enzyme. After incubation of BNeV VLPs at 50 μg/ml, the binding of BNeV VLPs was determined using hyperimmune serum against BNeV capsid protein, followed by addition of horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. The signal intensities for graphs in panels A and B were visualized using TMB at 450 nm in three independent experiments. Error bars indicate SD from triplicate samples.

    Article Snippet: The pFastBac1 donor plasmid was transformed into DH10Bac Escherichia coli , and its resultant recombinant bacmid DNA was transfected into Sf9 cells using Cellfectin II reagent (by following the manufacturer's instructions; Invitrogen).

    Techniques: Binding Assay, Blocking Assay, Infection, Incubation

    Electron micrograph of BNeV VLPs from and detection of BNeV capsid protein in rMA415-infected insect cells by immunofluorescence assay. (A) The rMA415 VLPs were purified by CsCl gradients from the cell culture supernatants of rMA415-infected Sf9 cells and visualized by negative staining with 3% phosphotungstic acid (pH 7.0). The panel on the right is a magnification of the panel on the left. The scale bars in the left and right panels correspond to 100 nm and 50 nm, respectively. (B) Sf9 cells were mock infected (left) or infected with rMA415. After 48 (middle) and 72 (right) h postinfection, cells were immunostained using the rabbit hyperimmune serum against BNeV capsid protein and FITC-conjugated goat anti-rabbit IgG antibody. The scale bars for left and right panels correspond to 50 μm.

    Journal: Journal of Virology

    Article Title: Bovine Nebovirus Interacts with a Wide Spectrum of Histo-Blood Group Antigens

    doi: 10.1128/JVI.02160-17

    Figure Lengend Snippet: Electron micrograph of BNeV VLPs from and detection of BNeV capsid protein in rMA415-infected insect cells by immunofluorescence assay. (A) The rMA415 VLPs were purified by CsCl gradients from the cell culture supernatants of rMA415-infected Sf9 cells and visualized by negative staining with 3% phosphotungstic acid (pH 7.0). The panel on the right is a magnification of the panel on the left. The scale bars in the left and right panels correspond to 100 nm and 50 nm, respectively. (B) Sf9 cells were mock infected (left) or infected with rMA415. After 48 (middle) and 72 (right) h postinfection, cells were immunostained using the rabbit hyperimmune serum against BNeV capsid protein and FITC-conjugated goat anti-rabbit IgG antibody. The scale bars for left and right panels correspond to 50 μm.

    Article Snippet: The pFastBac1 donor plasmid was transformed into DH10Bac Escherichia coli , and its resultant recombinant bacmid DNA was transfected into Sf9 cells using Cellfectin II reagent (by following the manufacturer's instructions; Invitrogen).

    Techniques: Infection, Immunofluorescence, Purification, Cell Culture, Negative Staining