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  • 99
    New England Biolabs ecor i
    Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with <t>EcoR</t> I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.
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    Thermo Fisher topo ta cloning kit
    <t>Topo</t> II and phospho-H3 are not essential for <t>rDNA</t> condensation. CH325 ( top2-4 ), JHY90 ( WT ), JHY91 ( H3-S10A ), and JHY93 ( H3-S10,28A ) cultures were synchronized in G1, and released into a Nz block either at 37°C (top2-4) or 23°C. After rearrest in M phase, cells were fixed and processed for rDNA FISH. rDNA loops were scored as condensed. Greater than 100 nuclei/sample were scored. Data for the condensins is from Fig. 2 and is shown for comparison.
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    TaKaRa ecori
    (a) Disruption of asp - orf2 using suicide vector pXAC-5528 (Δ asp ). The first homologous recombination produced a mutant 288 possessing asp - orf2 and the defective gene on pXAC-5528 (Δ asp ), and CAT and sacB genes. The second homologous recombination occurred between both types of genes located in tandem and produced the asp - orf2 -disrupted strain. (b) Southern-hybridization analysis of asp - orf2 . The asp - orf2 detection <t>DNA</t> probe (b, horizontal bar) that had <t>EcoRI</t> digestion sites at both sides was amplified using two primers AP-20 (5′-CATCGGCGGCAACCGCGGAA-3′) and AP-25 (5′-ATGCCGCTCTCCTTGCCGGT-3′), and labeled digoxigenin DNA Labeling Kit. Total DNAs extracted from both 288 (lanes 1 and 3) and 288 (Δ asp ) (lanes 2 and 4) using Qiagen Genomic-tips were digested with EcoRI and separated on 0.7% agarose gel. Southern-hybridization reaction with the digoxigenin-labeled probe was performed and hybridized fragments were detected with the digoxigenin luminescent detection kit. Numbers along the left side indicate DNA sizes in base pairs (bp). (C) SDS-PAGE of ASP. ASP (0.6 μg) was analyzed using a SDS-polyacrylamide gel (10%) in the presence (lane 3) or absence of 2-ME (lane 2), and the gel was silver-stained. Lane 1, molecular size markers.
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    Thermo Fisher ecor i
    Southern blot analysis of T 4 progenies of line IO6-97. Stable integration of RGA2 intron was detected in transgenic rice plants, no hybridization signal was observed in the respective non-transgenic control. Each lane consists of 10 µg genomic <t>DNA,</t> digested with <t>EcoR</t> I or Hind III. The position and sizes of markers are indicated (NT = Non-transgenic control, E = EcoR I and H = Hind III).
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    Millipore ecori
    Identification and characterization of <t>pMD18-T/gC</t> and pET32a-gC with restriction enzyme and PCR-based amplification. (a) Identification of pMD18-T/gC with restriction enzyme and PCR-based amplification. Lanes: 1, pMD18-T/gC digested with <t>EcoRI</t> and XhoI; 2, product amplified from pMD18-T/gC. M, DNA marker. (b) Characterization of the recombinant plasmid pET32a-gC by restriction digestion and PCR-based amplification. Lanes: 1, pET32a-gC digested with EcoRI and XhoI; 2, product amplified from pET32a-gC. M, DNA marker, marker III.
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    TaKaRa bamh i
    Identification and characterization of <t>pMD18-T/gC</t> and pET32a-gC with restriction enzyme and PCR-based amplification. (a) Identification of pMD18-T/gC with restriction enzyme and PCR-based amplification. Lanes: 1, pMD18-T/gC digested with <t>EcoRI</t> and XhoI; 2, product amplified from pMD18-T/gC. M, DNA marker. (b) Characterization of the recombinant plasmid pET32a-gC by restriction digestion and PCR-based amplification. Lanes: 1, pET32a-gC digested with EcoRI and XhoI; 2, product amplified from pET32a-gC. M, DNA marker, marker III.
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    Thermo Fisher bamh i
    Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the <t>BamH</t> I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.
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    TaKaRa xho i
    Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the <t>BamH</t> I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.
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    New England Biolabs t4 dna ligase
    Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the <t>BamH</t> I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.
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    Thermo Fisher xho i
    Southern blot analysis showing integration of the crtI (3.28 kb) and pmi (1.1 kb) gene in anther culture-derived plants. Stable integration of 3.28 kb EcoR I restriction fragment corresponding to the crtI gene and 1.1 kb <t>Xho</t> I restriction fragment corresponding to the pmi gene in different plant lines. No hybridization signal was observed in non-transgenic control plants. 10 µg of <t>DNA</t> was digested and it was separated on a 1% TAE-agarose gel.
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    Thermo Fisher kpn i
    Southern blot analysis showing integration of the crtI (3.28 kb) and pmi (1.1 kb) gene in anther culture-derived plants. Stable integration of 3.28 kb EcoR I restriction fragment corresponding to the crtI gene and 1.1 kb <t>Xho</t> I restriction fragment corresponding to the pmi gene in different plant lines. No hybridization signal was observed in non-transgenic control plants. 10 µg of <t>DNA</t> was digested and it was separated on a 1% TAE-agarose gel.
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    Thermo Fisher t4 dna ligase
    Southern blot analysis showing integration of the crtI (3.28 kb) and pmi (1.1 kb) gene in anther culture-derived plants. Stable integration of 3.28 kb EcoR I restriction fragment corresponding to the crtI gene and 1.1 kb <t>Xho</t> I restriction fragment corresponding to the pmi gene in different plant lines. No hybridization signal was observed in non-transgenic control plants. 10 µg of <t>DNA</t> was digested and it was separated on a 1% TAE-agarose gel.
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    TaKaRa bgl ii
    The confirmations of transformed E. coli carrying pEGFP-env-tm JDV. Selection bacteria in LB agar with Kanamycin 50 μg/ml: A – The confirmation of two selected clones using colony-PCR. Untransformed bacteria (control −), plasmid with env-tm gene (control +) and two colonies that were randomly picked from selection media (clones 1 and 2). B – Restriction analysis using <t>EcoR</t> I and <t>Bgl</t> II with undigested plasmid (uncut) and digested plasmid (cut)
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    Millipore xho
    The confirmations of transformed E. coli carrying pEGFP-env-tm JDV. Selection bacteria in LB agar with Kanamycin 50 μg/ml: A – The confirmation of two selected clones using colony-PCR. Untransformed bacteria (control −), plasmid with env-tm gene (control +) and two colonies that were randomly picked from selection media (clones 1 and 2). B – Restriction analysis using <t>EcoR</t> I and <t>Bgl</t> II with undigested plasmid (uncut) and digested plasmid (cut)
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    Thermo Fisher zero blunt pcr cloning kit
    Mutagenesis of <t>p48</t> catalytic residues in the CHV-1/EP713 infectious cDNA clone. (A) Colony morphology of EP155 and Δ dcl2 strains transfected with RNA transcripts corresponding to wild-type CHV-1/EP713 and p48 protease mutant viruses CHV-1/p48(C341S), CHV-1/p48(H388S), and CHV-1/p48(C341S:H388S). Infection of strain EP155 with CHV-1/p48(C341S) produced a fungal phenotype similar to CHV-1/EP713, including a white phenotype and a reduction in sporulation. Infection of EP155 with CHV-1/p48(H388S) and CHV-1/p48(C341S:H388S) resulted in further reduction of aerial hyphae; however, there was increased fungal pigmentation compared to CHV-1/EP713 infection. Infection of the Δ dcl2 strain with the p48 protease mutant viruses produced a phenotype similar to that of CHV-1/EP713-infected colonies. (B) Real-time <t>RT-PCR</t> quantification of viral RNA accumulation for p48 catalytic residue mutant viruses. Viral RNA accumulation levels were measured as described in Materials and Methods and are reported as percentages of the value obtained for CHV-1/EP713-infected EP155 fungal extracts, with standard deviations indicated by the error bars, based on three replicas each for three independent infected colonies cultured in parallel.
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    New England Biolabs bamh i
    Design of shRNA oligonucleotides for knocking down gene expression and silent mutations for knocking-in gene expression at internal gene regions. In A), annealed shRNA oligonucleotides form dsDNA fragment with BamH I/ Hind <t>III</t> sites, respectively (shown
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    TaKaRa xba i
    Electrophoresis of PCR product and pUCm-T-ZNRD1 digested with enzymes. Lane 1: PCR product; Lane 2, 3: Marker (200 bp); Lane 4: recombinant of pUCm-T-ZNRD1 cleaved by <t>EcoR</t> V and Xba I; Lane 5: recombinant of pUCm-T-ZNRD1.
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    Thermo Fisher xba i
    Electrophoresis of PCR product and pUCm-T-ZNRD1 digested with enzymes. Lane 1: PCR product; Lane 2, 3: Marker (200 bp); Lane 4: recombinant of pUCm-T-ZNRD1 cleaved by <t>EcoR</t> V and Xba I; Lane 5: recombinant of pUCm-T-ZNRD1.
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    Thermo Fisher pcdna3 1 vector
    NAC inhibits migration and invasion of GBM cells by suppressing Notch2 pathway. a Migration rate was measured by wound healing assay. Scale bar: 500 μm. b Transwell invasion assays of U87 and U251 cells. U87 and U251 cells were electroporated with <t>pcDNA3.1-Notch2</t> or pcDNA3.1-EV, pcDNA3.1-EV served as a control, followed by BSO (1 mM, 6 h) and NAC (10 mM, 12 h) treatment. Scale bar: 200 μm. All data are presented as means ± SD of three independent experiments. * P
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    Image Search Results


    Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.

    Journal: International Journal of Molecular Sciences

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino

    doi: 10.3390/ijms17101690

    Figure Lengend Snippet: Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.

    Article Snippet: The digested products were ligated at 16 °C for 4 h. The ligation mixture contained the digested fragments, 10 pmol EcoR I and 100 pmol Mse I adapteors , and 4 U T4 DNA polymerase (NEB).

    Techniques: DNA Methylation Assay, Methylation

    Topo II and phospho-H3 are not essential for rDNA condensation. CH325 ( top2-4 ), JHY90 ( WT ), JHY91 ( H3-S10A ), and JHY93 ( H3-S10,28A ) cultures were synchronized in G1, and released into a Nz block either at 37°C (top2-4) or 23°C. After rearrest in M phase, cells were fixed and processed for rDNA FISH. rDNA loops were scored as condensed. Greater than 100 nuclei/sample were scored. Data for the condensins is from Fig. 2 and is shown for comparison.

    Journal: The Journal of Cell Biology

    Article Title: In vivo dissection of the chromosome condensation machinery

    doi: 10.1083/jcb.200109056

    Figure Lengend Snippet: Topo II and phospho-H3 are not essential for rDNA condensation. CH325 ( top2-4 ), JHY90 ( WT ), JHY91 ( H3-S10A ), and JHY93 ( H3-S10,28A ) cultures were synchronized in G1, and released into a Nz block either at 37°C (top2-4) or 23°C. After rearrest in M phase, cells were fixed and processed for rDNA FISH. rDNA loops were scored as condensed. Greater than 100 nuclei/sample were scored. Data for the condensins is from Fig. 2 and is shown for comparison.

    Article Snippet: PCR cloning was performed using the TOPO-TA cloning kit (Invitrogen). rDNA and CEN16 proximal FISH probes were generated as previously described ( ).

    Techniques: Blocking Assay, Fluorescence In Situ Hybridization

    (a) Disruption of asp - orf2 using suicide vector pXAC-5528 (Δ asp ). The first homologous recombination produced a mutant 288 possessing asp - orf2 and the defective gene on pXAC-5528 (Δ asp ), and CAT and sacB genes. The second homologous recombination occurred between both types of genes located in tandem and produced the asp - orf2 -disrupted strain. (b) Southern-hybridization analysis of asp - orf2 . The asp - orf2 detection DNA probe (b, horizontal bar) that had EcoRI digestion sites at both sides was amplified using two primers AP-20 (5′-CATCGGCGGCAACCGCGGAA-3′) and AP-25 (5′-ATGCCGCTCTCCTTGCCGGT-3′), and labeled digoxigenin DNA Labeling Kit. Total DNAs extracted from both 288 (lanes 1 and 3) and 288 (Δ asp ) (lanes 2 and 4) using Qiagen Genomic-tips were digested with EcoRI and separated on 0.7% agarose gel. Southern-hybridization reaction with the digoxigenin-labeled probe was performed and hybridized fragments were detected with the digoxigenin luminescent detection kit. Numbers along the left side indicate DNA sizes in base pairs (bp). (C) SDS-PAGE of ASP. ASP (0.6 μg) was analyzed using a SDS-polyacrylamide gel (10%) in the presence (lane 3) or absence of 2-ME (lane 2), and the gel was silver-stained. Lane 1, molecular size markers.

    Journal: Fems Microbiology Letters

    Article Title: Impaired plasma clottability induction through fibrinogen degradation by ASP, a serine protease released from Aeromonas sobria

    doi: 10.1111/j.1574-6968.2008.01184.x

    Figure Lengend Snippet: (a) Disruption of asp - orf2 using suicide vector pXAC-5528 (Δ asp ). The first homologous recombination produced a mutant 288 possessing asp - orf2 and the defective gene on pXAC-5528 (Δ asp ), and CAT and sacB genes. The second homologous recombination occurred between both types of genes located in tandem and produced the asp - orf2 -disrupted strain. (b) Southern-hybridization analysis of asp - orf2 . The asp - orf2 detection DNA probe (b, horizontal bar) that had EcoRI digestion sites at both sides was amplified using two primers AP-20 (5′-CATCGGCGGCAACCGCGGAA-3′) and AP-25 (5′-ATGCCGCTCTCCTTGCCGGT-3′), and labeled digoxigenin DNA Labeling Kit. Total DNAs extracted from both 288 (lanes 1 and 3) and 288 (Δ asp ) (lanes 2 and 4) using Qiagen Genomic-tips were digested with EcoRI and separated on 0.7% agarose gel. Southern-hybridization reaction with the digoxigenin-labeled probe was performed and hybridized fragments were detected with the digoxigenin luminescent detection kit. Numbers along the left side indicate DNA sizes in base pairs (bp). (C) SDS-PAGE of ASP. ASP (0.6 μg) was analyzed using a SDS-polyacrylamide gel (10%) in the presence (lane 3) or absence of 2-ME (lane 2), and the gel was silver-stained. Lane 1, molecular size markers.

    Article Snippet: Preparation of asp -disrupted or -introduced strain Fragments of strain 288 chromosomal DNA digested with the EcoRI were inserted into the EcoRI site of pUC119 ( ) and introduced into E. coli HB101 (TaKaRa Co., Kyoto, Japan).

    Techniques: Plasmid Preparation, Homologous Recombination, Produced, Mutagenesis, Hybridization, Amplification, Labeling, DNA Labeling, Agarose Gel Electrophoresis, SDS Page, Staining

    Identification of the recombinant plasmid. (A) Detection of the housekeeping gene GAPDH. (B) Identification of the target gene by RT-PCR. (C) PCR product of the Fyn gene was subcloned into pMD18-T-Fyn and pEGFP-N1-Fyn. (D) Identification of pMD18-T-Fyn fragments produced by restriction enzyme digestion with Eco R I and Sma I. (E) Identification of pEGFP-N1-Fyn fragments generated by restriction enzyme digestion with Eco R I and Sma I. Lane 1, RT-PCR GAPDH product; Lane 2, negative control; Lane 3, RT-PCR Fyn product from brain; Lane 4, positive control; Lane 5, PCR product of pMD18-T-Fyn; Lane 6, PCR product of pEGFP-N1-Fyn; Lanes 7 and 9, positive control; Lanes 8 and 10, recombinant plasmid identification by digestion with EcoR I and Sma I (pMD18-T-Fyn, 2700 bp; pEGFP-N1, 4700 bp; Fyn, 1611 bp); Lane M, DNA marker.

    Journal: Journal of Veterinary Science

    Article Title: Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells

    doi: 10.4142/jvs.2014.15.1.111

    Figure Lengend Snippet: Identification of the recombinant plasmid. (A) Detection of the housekeeping gene GAPDH. (B) Identification of the target gene by RT-PCR. (C) PCR product of the Fyn gene was subcloned into pMD18-T-Fyn and pEGFP-N1-Fyn. (D) Identification of pMD18-T-Fyn fragments produced by restriction enzyme digestion with Eco R I and Sma I. (E) Identification of pEGFP-N1-Fyn fragments generated by restriction enzyme digestion with Eco R I and Sma I. Lane 1, RT-PCR GAPDH product; Lane 2, negative control; Lane 3, RT-PCR Fyn product from brain; Lane 4, positive control; Lane 5, PCR product of pMD18-T-Fyn; Lane 6, PCR product of pEGFP-N1-Fyn; Lanes 7 and 9, positive control; Lanes 8 and 10, recombinant plasmid identification by digestion with EcoR I and Sma I (pMD18-T-Fyn, 2700 bp; pEGFP-N1, 4700 bp; Fyn, 1611 bp); Lane M, DNA marker.

    Article Snippet: The PCR product was digested with EcoR I and Sma I, and ligated into a pEGFP-N1 vector (Clontech; Takara Bio) to produce the recombinant expression vector pEGFP-N1-Fyn.

    Techniques: Recombinant, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Produced, Generated, Negative Control, Positive Control, Marker

    Generation of Tg( MYCN :HSE:EGFP) zebrafish line. (A) Schematic diagram of the structure of PSGH2/MYCN recombinant plasmid. A mouse-MYCN fragment was cloned from HA-MYCN plasmid and inserted into the EcoRI and EcoRV sites of the PSGH2 vector. (B) A schematic presentation of the heat shock element (HSE) promoter. The artificial promoter contains eight multimerized heat shock elements flanked by two minimal promoters in opposed orientation (black arrowhead). EGFP and MYCN are expressed from the bidirectional promoter. The vector is flanked by I-SceI meganuclease sites (arrows). pA, SV40 polyadenylation signal. (C) Transgenic verification by qRT-PCR: M: TAKARA DL2000 marker; lane 1: Blank control (double distilled water); lane 2 and 3: WT and Tg F1 generation embryos at 3 dpf, respectively; lane 4: Positive control (plasmid). (D) Transgenic verification by westernblot: lane 1: WT embryo at 3 dpf; lane 2 and 3: Tg F1 and F2 generation embryos at 3 dpf, respectively. (E–F) EGFP (+) F0 mosaic zebrafish at 24 hours (×50) and 60 days post microinjection (×7.5). (G–H) EGFP (+) F1 Tg zebrafish at 24 hpf (×50) and 60 dpf (×10). (I) Expression of total MYCN (murine exogenous and zebrafish endogenous expression), which was increased gradually in Tg F1, F2 generation fish comparing with that in WT. (J) Expression of NDRG1 , which is negative controlled by MYCN in human, was keeping a low lever in Tg F1 and F2 generation. **, P

    Journal: PLoS ONE

    Article Title: MYCN Transgenic Zebrafish Model with the Characterization of Acute Myeloid Leukemia and Altered Hematopoiesis

    doi: 10.1371/journal.pone.0059070

    Figure Lengend Snippet: Generation of Tg( MYCN :HSE:EGFP) zebrafish line. (A) Schematic diagram of the structure of PSGH2/MYCN recombinant plasmid. A mouse-MYCN fragment was cloned from HA-MYCN plasmid and inserted into the EcoRI and EcoRV sites of the PSGH2 vector. (B) A schematic presentation of the heat shock element (HSE) promoter. The artificial promoter contains eight multimerized heat shock elements flanked by two minimal promoters in opposed orientation (black arrowhead). EGFP and MYCN are expressed from the bidirectional promoter. The vector is flanked by I-SceI meganuclease sites (arrows). pA, SV40 polyadenylation signal. (C) Transgenic verification by qRT-PCR: M: TAKARA DL2000 marker; lane 1: Blank control (double distilled water); lane 2 and 3: WT and Tg F1 generation embryos at 3 dpf, respectively; lane 4: Positive control (plasmid). (D) Transgenic verification by westernblot: lane 1: WT embryo at 3 dpf; lane 2 and 3: Tg F1 and F2 generation embryos at 3 dpf, respectively. (E–F) EGFP (+) F0 mosaic zebrafish at 24 hours (×50) and 60 days post microinjection (×7.5). (G–H) EGFP (+) F1 Tg zebrafish at 24 hpf (×50) and 60 dpf (×10). (I) Expression of total MYCN (murine exogenous and zebrafish endogenous expression), which was increased gradually in Tg F1, F2 generation fish comparing with that in WT. (J) Expression of NDRG1 , which is negative controlled by MYCN in human, was keeping a low lever in Tg F1 and F2 generation. **, P

    Article Snippet: Construction of PSGH2/MYCN plasmid A mouse-MYCN fragment was extracted from HA-MYCN plasmid and subcloned into the EcoRI and EcoRV (Takara, Japan) sites of the PSGH2 vector to obtain the mMYCN -HSE-EGFP construct ( , ).

    Techniques: Recombinant, Plasmid Preparation, Clone Assay, Transgenic Assay, Quantitative RT-PCR, Marker, Positive Control, Expressing, Fluorescence In Situ Hybridization

    Southern blot analysis of T 4 progenies of line IO6-97. Stable integration of RGA2 intron was detected in transgenic rice plants, no hybridization signal was observed in the respective non-transgenic control. Each lane consists of 10 µg genomic DNA, digested with EcoR I or Hind III. The position and sizes of markers are indicated (NT = Non-transgenic control, E = EcoR I and H = Hind III).

    Journal: PLoS ONE

    Article Title: Development of Low Phytate Rice by RNAi Mediated Seed-Specific Silencing of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase Gene (IPK1)

    doi: 10.1371/journal.pone.0068161

    Figure Lengend Snippet: Southern blot analysis of T 4 progenies of line IO6-97. Stable integration of RGA2 intron was detected in transgenic rice plants, no hybridization signal was observed in the respective non-transgenic control. Each lane consists of 10 µg genomic DNA, digested with EcoR I or Hind III. The position and sizes of markers are indicated (NT = Non-transgenic control, E = EcoR I and H = Hind III).

    Article Snippet: Genomic DNA (10 µg) was digested separately with EcoR I and Hind III (Fermentas), separated on a 1% agarose gel, and transferred to a nylon membrane (Hybond N+, Amersham, GE Healthcare).

    Techniques: Southern Blot, Transgenic Assay, Hybridization

    Identification and characterization of pMD18-T/gC and pET32a-gC with restriction enzyme and PCR-based amplification. (a) Identification of pMD18-T/gC with restriction enzyme and PCR-based amplification. Lanes: 1, pMD18-T/gC digested with EcoRI and XhoI; 2, product amplified from pMD18-T/gC. M, DNA marker. (b) Characterization of the recombinant plasmid pET32a-gC by restriction digestion and PCR-based amplification. Lanes: 1, pET32a-gC digested with EcoRI and XhoI; 2, product amplified from pET32a-gC. M, DNA marker, marker III.

    Journal: Virology Journal

    Article Title: Identification and characterization of duck plague virus glycoprotein C gene and gene product

    doi: 10.1186/1743-422X-7-349

    Figure Lengend Snippet: Identification and characterization of pMD18-T/gC and pET32a-gC with restriction enzyme and PCR-based amplification. (a) Identification of pMD18-T/gC with restriction enzyme and PCR-based amplification. Lanes: 1, pMD18-T/gC digested with EcoRI and XhoI; 2, product amplified from pMD18-T/gC. M, DNA marker. (b) Characterization of the recombinant plasmid pET32a-gC by restriction digestion and PCR-based amplification. Lanes: 1, pET32a-gC digested with EcoRI and XhoI; 2, product amplified from pET32a-gC. M, DNA marker, marker III.

    Article Snippet: After verified by PCR, restriction analysis and DNA sequencing (TaKaRa), the gC gene fragment, which was obtained by digestion of pMD18-T/gC with EcoRI and XhoI, was ligated into prokaryotic vector pET32a(+) (Novagen) (Figure ), which was digested previously with the same restriction enzymes.

    Techniques: Polymerase Chain Reaction, Amplification, Marker, Recombinant, Plasmid Preparation

    Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the BamH I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.

    Journal: Virology

    Article Title: Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs

    doi: 10.1016/j.virol.2010.03.025

    Figure Lengend Snippet: Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the BamH I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.

    Article Snippet: It was generated by digesting with BamH I and then filling in the sticky ends with DNA polymerase Pfx (Invitrogen) followed by blunt-end re-ligation. pGlucG was derived by inserting the PCR-amplified 12 nt 5′UTR of GRV into EcoR I/Sca I-digested pTUlucTU, in place of the TBTV 5′ UTR.

    Techniques: Luciferase, Activity Assay, Generated, Mutagenesis, Construct, Sequencing

    ). 17 nt CS is shown in bold italic. (B) Genome organization of RSDaV RNA and maps of translation reporter constructs. RSlucRS has both UTRs of RSDaV flanking the luciferase coding sequence (fLUC). RSluc has only the RSDaV 5′UTR. RSlucRSBF differs from RSlucRS only by a 4 nt GAUC insertion (underlined) in the BamH I site. Bases are numbered according to their position in the RSDaV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities are normalized to uncapped RSlucRS (defined as 100%). Error bars indicate standard error.

    Journal: Virology

    Article Title: Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs

    doi: 10.1016/j.virol.2010.03.025

    Figure Lengend Snippet: ). 17 nt CS is shown in bold italic. (B) Genome organization of RSDaV RNA and maps of translation reporter constructs. RSlucRS has both UTRs of RSDaV flanking the luciferase coding sequence (fLUC). RSluc has only the RSDaV 5′UTR. RSlucRSBF differs from RSlucRS only by a 4 nt GAUC insertion (underlined) in the BamH I site. Bases are numbered according to their position in the RSDaV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities are normalized to uncapped RSlucRS (defined as 100%). Error bars indicate standard error.

    Article Snippet: It was generated by digesting with BamH I and then filling in the sticky ends with DNA polymerase Pfx (Invitrogen) followed by blunt-end re-ligation. pGlucG was derived by inserting the PCR-amplified 12 nt 5′UTR of GRV into EcoR I/Sca I-digested pTUlucTU, in place of the TBTV 5′ UTR.

    Techniques: Construct, Luciferase, Sequencing

    Southern blot analysis showing integration of the crtI (3.28 kb) and pmi (1.1 kb) gene in anther culture-derived plants. Stable integration of 3.28 kb EcoR I restriction fragment corresponding to the crtI gene and 1.1 kb Xho I restriction fragment corresponding to the pmi gene in different plant lines. No hybridization signal was observed in non-transgenic control plants. 10 µg of DNA was digested and it was separated on a 1% TAE-agarose gel.

    Journal: PLoS ONE

    Article Title: Genetic Stability Developed for ?-Carotene Synthesis in BR29 Rice Line Using Dihaploid Homozygosity

    doi: 10.1371/journal.pone.0100212

    Figure Lengend Snippet: Southern blot analysis showing integration of the crtI (3.28 kb) and pmi (1.1 kb) gene in anther culture-derived plants. Stable integration of 3.28 kb EcoR I restriction fragment corresponding to the crtI gene and 1.1 kb Xho I restriction fragment corresponding to the pmi gene in different plant lines. No hybridization signal was observed in non-transgenic control plants. 10 µg of DNA was digested and it was separated on a 1% TAE-agarose gel.

    Article Snippet: Next, 10 µg of DNA was digested using restriction endonucleases, EcoR I for crtI , and Xho I for pmi gene (Invitrogen, CA) and the digested DNA was separated on a 1% (w/v) TAE-agarose gel.

    Techniques: Southern Blot, Derivative Assay, Hybridization, Transgenic Assay, Agarose Gel Electrophoresis

    The confirmations of transformed E. coli carrying pEGFP-env-tm JDV. Selection bacteria in LB agar with Kanamycin 50 μg/ml: A – The confirmation of two selected clones using colony-PCR. Untransformed bacteria (control −), plasmid with env-tm gene (control +) and two colonies that were randomly picked from selection media (clones 1 and 2). B – Restriction analysis using EcoR I and Bgl II with undigested plasmid (uncut) and digested plasmid (cut)

    Journal: Journal of Veterinary Research

    Article Title: In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate

    doi: 10.2478/jvetres-2019-0018

    Figure Lengend Snippet: The confirmations of transformed E. coli carrying pEGFP-env-tm JDV. Selection bacteria in LB agar with Kanamycin 50 μg/ml: A – The confirmation of two selected clones using colony-PCR. Untransformed bacteria (control −), plasmid with env-tm gene (control +) and two colonies that were randomly picked from selection media (clones 1 and 2). B – Restriction analysis using EcoR I and Bgl II with undigested plasmid (uncut) and digested plasmid (cut)

    Article Snippet: Then, the transgene was chemically synthesised with restriction sites of Bgl II and EcoR I and cloned into the pEGFP-C1 vector (Clontech) by Gene Universal Inc., resulting in pEGFP-env-tm JDV ( ).

    Techniques: Transformation Assay, Selection, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation

    Mutagenesis of p48 catalytic residues in the CHV-1/EP713 infectious cDNA clone. (A) Colony morphology of EP155 and Δ dcl2 strains transfected with RNA transcripts corresponding to wild-type CHV-1/EP713 and p48 protease mutant viruses CHV-1/p48(C341S), CHV-1/p48(H388S), and CHV-1/p48(C341S:H388S). Infection of strain EP155 with CHV-1/p48(C341S) produced a fungal phenotype similar to CHV-1/EP713, including a white phenotype and a reduction in sporulation. Infection of EP155 with CHV-1/p48(H388S) and CHV-1/p48(C341S:H388S) resulted in further reduction of aerial hyphae; however, there was increased fungal pigmentation compared to CHV-1/EP713 infection. Infection of the Δ dcl2 strain with the p48 protease mutant viruses produced a phenotype similar to that of CHV-1/EP713-infected colonies. (B) Real-time RT-PCR quantification of viral RNA accumulation for p48 catalytic residue mutant viruses. Viral RNA accumulation levels were measured as described in Materials and Methods and are reported as percentages of the value obtained for CHV-1/EP713-infected EP155 fungal extracts, with standard deviations indicated by the error bars, based on three replicas each for three independent infected colonies cultured in parallel.

    Journal: Journal of Virology

    Article Title: Mutagenesis of the Catalytic and Cleavage Site Residues of the Hypovirus Papain-Like Proteases p29 and p48 Reveals Alternative Processing and Contributions to Optimal Viral RNA Accumulation

    doi: 10.1128/JVI.01489-14

    Figure Lengend Snippet: Mutagenesis of p48 catalytic residues in the CHV-1/EP713 infectious cDNA clone. (A) Colony morphology of EP155 and Δ dcl2 strains transfected with RNA transcripts corresponding to wild-type CHV-1/EP713 and p48 protease mutant viruses CHV-1/p48(C341S), CHV-1/p48(H388S), and CHV-1/p48(C341S:H388S). Infection of strain EP155 with CHV-1/p48(C341S) produced a fungal phenotype similar to CHV-1/EP713, including a white phenotype and a reduction in sporulation. Infection of EP155 with CHV-1/p48(H388S) and CHV-1/p48(C341S:H388S) resulted in further reduction of aerial hyphae; however, there was increased fungal pigmentation compared to CHV-1/EP713 infection. Infection of the Δ dcl2 strain with the p48 protease mutant viruses produced a phenotype similar to that of CHV-1/EP713-infected colonies. (B) Real-time RT-PCR quantification of viral RNA accumulation for p48 catalytic residue mutant viruses. Viral RNA accumulation levels were measured as described in Materials and Methods and are reported as percentages of the value obtained for CHV-1/EP713-infected EP155 fungal extracts, with standard deviations indicated by the error bars, based on three replicas each for three independent infected colonies cultured in parallel.

    Article Snippet: The two fragments were stitched together by a second round of PCR using the primers AscI-F and 4KR to create the final Δp29-p48 triple mutant fragment, which was subsequently cloned into a PCR-blunt vector (Zero-Blunt PCR cloning kit; Invitrogen, Carlsbad, CA).

    Techniques: Mutagenesis, Transfection, Infection, Produced, Quantitative RT-PCR, Cell Culture

    Design of shRNA oligonucleotides for knocking down gene expression and silent mutations for knocking-in gene expression at internal gene regions. In A), annealed shRNA oligonucleotides form dsDNA fragment with BamH I/ Hind III sites, respectively (shown

    Journal: Nature protocols

    Article Title: Simultaneous knockdown of the expression of two genes using multiple shRNAs and subsequent knock-in of their expression

    doi: 10.1038/nprot.2009.145

    Figure Lengend Snippet: Design of shRNA oligonucleotides for knocking down gene expression and silent mutations for knocking-in gene expression at internal gene regions. In A), annealed shRNA oligonucleotides form dsDNA fragment with BamH I/ Hind III sites, respectively (shown

    Article Snippet: Hygromycin B (Cellgro, cat. no. 30-240-CR) Hind III, EcoR I, Xho I, Mfe I, BamH I, Dpn I (New England BioLabs, cat. no. R0104S, R0101S, R0146S, R0589S, R0136S, R0176S, respectively) or other suitable restriction enzymes.

    Techniques: shRNA, Expressing

    Electrophoresis of PCR product and pUCm-T-ZNRD1 digested with enzymes. Lane 1: PCR product; Lane 2, 3: Marker (200 bp); Lane 4: recombinant of pUCm-T-ZNRD1 cleaved by EcoR V and Xba I; Lane 5: recombinant of pUCm-T-ZNRD1.

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Effect of ZNRD1 gene antisense RNA on drug resistant gastric cancer cells

    doi: 10.3748/wjg.v9.i5.894

    Figure Lengend Snippet: Electrophoresis of PCR product and pUCm-T-ZNRD1 digested with enzymes. Lane 1: PCR product; Lane 2, 3: Marker (200 bp); Lane 4: recombinant of pUCm-T-ZNRD1 cleaved by EcoR V and Xba I; Lane 5: recombinant of pUCm-T-ZNRD1.

    Article Snippet: EcoR V, Xba I, BamH I, cloning vector pUCm-T and T4 DNA ligase were purchased from Takara; MTT, DEPC from Sigma.

    Techniques: Electrophoresis, Polymerase Chain Reaction, Marker, Recombinant

    NAC inhibits migration and invasion of GBM cells by suppressing Notch2 pathway. a Migration rate was measured by wound healing assay. Scale bar: 500 μm. b Transwell invasion assays of U87 and U251 cells. U87 and U251 cells were electroporated with pcDNA3.1-Notch2 or pcDNA3.1-EV, pcDNA3.1-EV served as a control, followed by BSO (1 mM, 6 h) and NAC (10 mM, 12 h) treatment. Scale bar: 200 μm. All data are presented as means ± SD of three independent experiments. * P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: N-acetylcysteine decreases malignant characteristics of glioblastoma cells by inhibiting Notch2 signaling

    doi: 10.1186/s13046-018-1016-8

    Figure Lengend Snippet: NAC inhibits migration and invasion of GBM cells by suppressing Notch2 pathway. a Migration rate was measured by wound healing assay. Scale bar: 500 μm. b Transwell invasion assays of U87 and U251 cells. U87 and U251 cells were electroporated with pcDNA3.1-Notch2 or pcDNA3.1-EV, pcDNA3.1-EV served as a control, followed by BSO (1 mM, 6 h) and NAC (10 mM, 12 h) treatment. Scale bar: 200 μm. All data are presented as means ± SD of three independent experiments. * P

    Article Snippet: The gel-purified PCR products were digested with the restriction enzymes, EcoR I and Not I (New England Biolabs), and cloned into the pcDNA3.1 vector (Invitrogen).

    Techniques: Migration, Wound Healing Assay

    NAC attenuates proliferation of GBM cells through Notch2 signaling. a ,Notch2 was analyzed by western blot. b and c Cell viability was analyzed by CCK8 at 450 nm. d and e The cell cycle analysis was measured by the percentage of cells in each phase in U87 and U251 cells. f The expression levels of P21, cyclin E and CDK2 in U87 and U251 cells. All cells were electroporated with pcDNA3.1-Notch2 or pcDNA3.1-EV, pcDNA3.1-EV served as a control, followed by BSO (1 mM, 12 h) and NAC (10 mM, 24 h) treatment. β-actin was used as a loading control. All data are presented as means ± SD of three independent experiments. * P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: N-acetylcysteine decreases malignant characteristics of glioblastoma cells by inhibiting Notch2 signaling

    doi: 10.1186/s13046-018-1016-8

    Figure Lengend Snippet: NAC attenuates proliferation of GBM cells through Notch2 signaling. a ,Notch2 was analyzed by western blot. b and c Cell viability was analyzed by CCK8 at 450 nm. d and e The cell cycle analysis was measured by the percentage of cells in each phase in U87 and U251 cells. f The expression levels of P21, cyclin E and CDK2 in U87 and U251 cells. All cells were electroporated with pcDNA3.1-Notch2 or pcDNA3.1-EV, pcDNA3.1-EV served as a control, followed by BSO (1 mM, 12 h) and NAC (10 mM, 24 h) treatment. β-actin was used as a loading control. All data are presented as means ± SD of three independent experiments. * P

    Article Snippet: The gel-purified PCR products were digested with the restriction enzymes, EcoR I and Not I (New England Biolabs), and cloned into the pcDNA3.1 vector (Invitrogen).

    Techniques: Western Blot, Cell Cycle Assay, Expressing