mammalian expression vector pcdna3 1  (Thermo Fisher)


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    Name:
    pcDNA 3 1 Mammalian Expression Vector
    Description:
    This pcDNA 3 1 vector is designed for high level constitutive expression in a variety of mammalian cell lines It contains a Geneticin selectable marker and a reverse orientation multiple cloning site The pcDNA 3 1 Expression Vector Family Three untagged versions of pcDNA 3 1 available separately each with a different selectable marker Geneticin Zeocin or Hygromycin are for use alone or in co transfections All three vectors offer the following features • Cytomegalovirus CMV enhancer promoter for high level expression• Large multiple cloning site in either forward or reverse orientations• Bovine Growth Hormone BGH polyadenylation signal and transcription termination sequence for enhanced mRNA stability• SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen i e COS 1 and COS 7 • Ampicillin resistance gene and pUC origin for selection and maintenance in E coli
    Catalog Number:
    v79520
    Price:
    None
    Applications:
    Constitutive Expression|Mammalian Expression|Protein Biology|Protein Expression
    Category:
    DNA Vectors Clones Purified Nucleic Acids Libraries
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    Structured Review

    Thermo Fisher mammalian expression vector pcdna3 1
    UBIAD1 inhibits the Ras/ERK signaling pathway. a UBIAD1 reduced cell viability in T24 bladder cancer cells. T24 cells were transiently transfected with <t>pcDNA3.1-UBIAD1</t> plasmid. Twenty-four hours after transfection, cell viability was detected by the MTT assay. *** p
    This pcDNA 3 1 vector is designed for high level constitutive expression in a variety of mammalian cell lines It contains a Geneticin selectable marker and a reverse orientation multiple cloning site The pcDNA 3 1 Expression Vector Family Three untagged versions of pcDNA 3 1 available separately each with a different selectable marker Geneticin Zeocin or Hygromycin are for use alone or in co transfections All three vectors offer the following features • Cytomegalovirus CMV enhancer promoter for high level expression• Large multiple cloning site in either forward or reverse orientations• Bovine Growth Hormone BGH polyadenylation signal and transcription termination sequence for enhanced mRNA stability• SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen i e COS 1 and COS 7 • Ampicillin resistance gene and pUC origin for selection and maintenance in E coli
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    Images

    1) Product Images from "UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras"

    Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1215-4

    UBIAD1 inhibits the Ras/ERK signaling pathway. a UBIAD1 reduced cell viability in T24 bladder cancer cells. T24 cells were transiently transfected with pcDNA3.1-UBIAD1 plasmid. Twenty-four hours after transfection, cell viability was detected by the MTT assay. *** p
    Figure Legend Snippet: UBIAD1 inhibits the Ras/ERK signaling pathway. a UBIAD1 reduced cell viability in T24 bladder cancer cells. T24 cells were transiently transfected with pcDNA3.1-UBIAD1 plasmid. Twenty-four hours after transfection, cell viability was detected by the MTT assay. *** p

    Techniques Used: Transfection, Plasmid Preparation, MTT Assay

    2) Product Images from "Vinexin β Interacts with Hepatitis C Virus NS5A, Modulating Its Hyperphosphorylation To Regulate Viral Propagation"

    Article Title: Vinexin β Interacts with Hepatitis C Virus NS5A, Modulating Its Hyperphosphorylation To Regulate Viral Propagation

    Journal: Journal of Virology

    doi: 10.1128/JVI.00567-15

    Vinexin β expression regulates the interaction of CK1α with NS5A. (A and B) HEK 293T cells were transfected with pRK-HA-vinexin β (HA-Vinβ), pcDNA3.1-NS5A (JFH1), and pRK-Flag-CK1α or pRK-Flag-CK1α (K46A).
    Figure Legend Snippet: Vinexin β expression regulates the interaction of CK1α with NS5A. (A and B) HEK 293T cells were transfected with pRK-HA-vinexin β (HA-Vinβ), pcDNA3.1-NS5A (JFH1), and pRK-Flag-CK1α or pRK-Flag-CK1α (K46A).

    Techniques Used: Expressing, Transfection

    3) Product Images from "Forced expression of heat-shock protein 70 increases the secretion of Hsp70 and provides protection against tumour growth"

    Article Title: Forced expression of heat-shock protein 70 increases the secretion of Hsp70 and provides protection against tumour growth

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601583

    ( A ) Western analysis for Hsp70 in supernatants of pcDNA3.1+Hsp70 and Renilla luciferase or mock-transfected TRAMP-C2 cells. Spent media were collected at 24, 48, and 72 h and concentrated as in Materials and methods. ( B ) Western analysis for Hsp70 in cell extracts of Hsp70, Renilla luciferase , and mock-transfected TRAMP-C2 cells. Ponseau S was used for normalisation. ( C ) Percent of Hsp70 in supernatants (Sup) and cell extracts (CE) of Hsp70 and mock-transfected TRAMP-C2 cells as determined by densitometry. ( D ). Comparison of luciferase activity in cell extracts (C) vs supernatants (S) in C2 cells transfected with pcDNA3.1+Hsp70 and Renilla luciferase at various time points. Spent media and whole-cell protein extracts were prepared as above. ( E ) Percentage of renilla protein was determined by luminescence.
    Figure Legend Snippet: ( A ) Western analysis for Hsp70 in supernatants of pcDNA3.1+Hsp70 and Renilla luciferase or mock-transfected TRAMP-C2 cells. Spent media were collected at 24, 48, and 72 h and concentrated as in Materials and methods. ( B ) Western analysis for Hsp70 in cell extracts of Hsp70, Renilla luciferase , and mock-transfected TRAMP-C2 cells. Ponseau S was used for normalisation. ( C ) Percent of Hsp70 in supernatants (Sup) and cell extracts (CE) of Hsp70 and mock-transfected TRAMP-C2 cells as determined by densitometry. ( D ). Comparison of luciferase activity in cell extracts (C) vs supernatants (S) in C2 cells transfected with pcDNA3.1+Hsp70 and Renilla luciferase at various time points. Spent media and whole-cell protein extracts were prepared as above. ( E ) Percentage of renilla protein was determined by luminescence.

    Techniques Used: Western Blot, Luciferase, Transfection, Activity Assay

    4) Product Images from "Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific"

    Article Title: Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific

    Journal: Asian Journal of Andrology

    doi: 10.1038/aja.2010.56

    Screening of stable clones expressing PSP94 by Western blot analysis. (A): Lanes 1–5 represent cell lysates from five different clones following PSP94 expression construct transfection of PC3 cells. Lane 6 is a cell lysate from one of the clones that was obtained following transfection with empty vector (pcDNA3.1+). The expression of recombinant PSP94HA was confirmed by probing the blot with anti-HA antibody. β-Actin was used as a loading control. (B): Western blot analysis of the TCA-precipitated proteins from the culture supernatants of stable clones PC3-A1v (which carried empty vector), PC3-A5 (which expressed PSP94HA) and PC3-D4 (which expressed PSP94HA) after roughly 30 passages in culture. Clones PC3-A5 and PC3-D4 stably expressed and secreted PSP94HA. The blot was probed with anti-HA antibody to detect PSP94HA, and with anti-β-actin antibody to show that the conditioned media were not contaminated with cellular contents.
    Figure Legend Snippet: Screening of stable clones expressing PSP94 by Western blot analysis. (A): Lanes 1–5 represent cell lysates from five different clones following PSP94 expression construct transfection of PC3 cells. Lane 6 is a cell lysate from one of the clones that was obtained following transfection with empty vector (pcDNA3.1+). The expression of recombinant PSP94HA was confirmed by probing the blot with anti-HA antibody. β-Actin was used as a loading control. (B): Western blot analysis of the TCA-precipitated proteins from the culture supernatants of stable clones PC3-A1v (which carried empty vector), PC3-A5 (which expressed PSP94HA) and PC3-D4 (which expressed PSP94HA) after roughly 30 passages in culture. Clones PC3-A5 and PC3-D4 stably expressed and secreted PSP94HA. The blot was probed with anti-HA antibody to detect PSP94HA, and with anti-β-actin antibody to show that the conditioned media were not contaminated with cellular contents.

    Techniques Used: Clone Assay, Expressing, Western Blot, Construct, Transfection, Plasmid Preparation, Recombinant, Stable Transfection

    Ectopic expression of PSP94 has an inhibitory effect on cell growth in a cell line specific manner. (A): A representative composite picture of the crystal violet staining from the clonogenic survival assay. A single well (of the six-well plate) is shown for each cell line (PC3, WPE1-NB26 and LNCaP) following transfection with empty vector, pcDNA3.1+ (left wells) or PSP94 expression construct (right wells). At 24 h after transfection, the cells were split and stable clones were selected on G418. Clones obtained after two weeks of selection were stained and counted. (B): Effect of the over-expression of PSP94 on the clonogenic survival of PC3, WPE1-NB26 and LNCaP cells. For each cell line, the number of clones obtained in the vector-transfected wells was considered to represent 100% survival. The number of clones obtained in the PSP94 expression construct-transfected wells was used to calculate the percentage of surviving cells with respect to the empty vector-transfected cells. The assay was repeated at least three times for each cell line. The graph is the average of three independent observations and is plotted as the mean percentage survival ± SE. The observed reduction in the percentage survival of WPE1-NB26 and LNCaP cells was statistically significant when compared with PC3 cells ( * P
    Figure Legend Snippet: Ectopic expression of PSP94 has an inhibitory effect on cell growth in a cell line specific manner. (A): A representative composite picture of the crystal violet staining from the clonogenic survival assay. A single well (of the six-well plate) is shown for each cell line (PC3, WPE1-NB26 and LNCaP) following transfection with empty vector, pcDNA3.1+ (left wells) or PSP94 expression construct (right wells). At 24 h after transfection, the cells were split and stable clones were selected on G418. Clones obtained after two weeks of selection were stained and counted. (B): Effect of the over-expression of PSP94 on the clonogenic survival of PC3, WPE1-NB26 and LNCaP cells. For each cell line, the number of clones obtained in the vector-transfected wells was considered to represent 100% survival. The number of clones obtained in the PSP94 expression construct-transfected wells was used to calculate the percentage of surviving cells with respect to the empty vector-transfected cells. The assay was repeated at least three times for each cell line. The graph is the average of three independent observations and is plotted as the mean percentage survival ± SE. The observed reduction in the percentage survival of WPE1-NB26 and LNCaP cells was statistically significant when compared with PC3 cells ( * P

    Techniques Used: Expressing, Staining, Clonogenic Cell Survival Assay, Transfection, Plasmid Preparation, Construct, Clone Assay, Selection, Over Expression

    5) Product Images from "Involvement of Daphnia pulicaria Sir2 in regulating stress response and lifespan"

    Article Title: Involvement of Daphnia pulicaria Sir2 in regulating stress response and lifespan

    Journal: Aging (Albany NY)

    doi:

    Daphnia Sir2 ORF produces a functional protein with catalytic activity (A) Expression of Daphnia Sir2 in Cos-1 cells . 100 ng each of Daphnia FLAG-Sir2/pcDNA3.1-, FLAG-TRBP/pcDNA3.1-, or empty vector pcDNA3.1- were transfected in Cos-1 cells using Effectene (Qiagen) and total protein extract was prepared 24 h after transfection. Western blot analysis was performed with anti-Flag antibody (M2, Sigma). Arrows indicate Flag-Sir2 and Flag-TRBP positions. (B) Sirt1 Activity Assay . Activity assay was performed using 5 μl of in vitro translated Daphnia Sir2. Human recombinant Sirt1 was used as a positive control. Ex527, a Sirt1/Sir2 specific inhibitor was used to ensure specificity that the observed deacetylase activity was that of Daphnia Sir2. Note that Daphnia Sir2 data represented is after subtracting the background activity obtained with unprimed (no plasmid DNA added) reticulocyte lysate. Error bars indicate standard deviations from 4 replicate assays. A Student's T Test was performed to determine statistical significance. The p values are as follows: *: 5.2×10 −7 (t stat: 22.2, df: 3); #: 4.8×10 −7 (t stat: 22.6, df: 3).
    Figure Legend Snippet: Daphnia Sir2 ORF produces a functional protein with catalytic activity (A) Expression of Daphnia Sir2 in Cos-1 cells . 100 ng each of Daphnia FLAG-Sir2/pcDNA3.1-, FLAG-TRBP/pcDNA3.1-, or empty vector pcDNA3.1- were transfected in Cos-1 cells using Effectene (Qiagen) and total protein extract was prepared 24 h after transfection. Western blot analysis was performed with anti-Flag antibody (M2, Sigma). Arrows indicate Flag-Sir2 and Flag-TRBP positions. (B) Sirt1 Activity Assay . Activity assay was performed using 5 μl of in vitro translated Daphnia Sir2. Human recombinant Sirt1 was used as a positive control. Ex527, a Sirt1/Sir2 specific inhibitor was used to ensure specificity that the observed deacetylase activity was that of Daphnia Sir2. Note that Daphnia Sir2 data represented is after subtracting the background activity obtained with unprimed (no plasmid DNA added) reticulocyte lysate. Error bars indicate standard deviations from 4 replicate assays. A Student's T Test was performed to determine statistical significance. The p values are as follows: *: 5.2×10 −7 (t stat: 22.2, df: 3); #: 4.8×10 −7 (t stat: 22.6, df: 3).

    Techniques Used: Functional Assay, Activity Assay, Expressing, Plasmid Preparation, Transfection, Western Blot, In Vitro, Recombinant, Positive Control, Histone Deacetylase Assay

    6) Product Images from "LINC00703 Acts as a Tumor Suppressor via Regulating miR-181a/KLF6 Axis in Gastric Cancer"

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

    Journal: Journal of Gastric Cancer

    doi: 10.5230/jgc.2019.19.e43

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

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

    7) Product Images from "Fractalkine Expression Induces Endothelial Progenitor Cell Lysis by Natural Killer Cells"

    Article Title: Fractalkine Expression Induces Endothelial Progenitor Cell Lysis by Natural Killer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026663

    Membrane FKN targets CD34 + -derived ECFC lysis by NK cells. ( A ) Cytotoxicity assay performed using PBMC effector cells isolated from 7 independent blood donors as described in Methods . Each independent assay was performed in triplicate (n = 7) and analysed using an effector: target ratio = 100∶1. ( B ) Representative illustration of ECFC lysis. CMFDA labeled target cells were analyzed by epi-fluorescence microscopy on an inverted microscope Nikon Eclipse TE 2000-U with a Plan Fluor 4x /0.13 objective, images were acquired using NIS elements AR software. ( C ) FKN enhances ECFC lysis by purified NK cells. CMFDA cytotoxicity assay performed using purified NK cells as effectors, effector: target ratio = 10∶1, (n = 3). ( D ) Addition of FKN neutralizing antibody reduces ECFC lysis. FKN-transfected ECFC were treated with 30 µg/ml of anti-human FKN antibody before addition of PBMC (n = 3). ( E ) Lysis of FKN expressing ECFC resulted in increased microparticles release after 24 h of incubation with PBMC.ECFC were labeled with the lipophilic DiD tracer and transfected with pCDNA3.1 vector coding for membrane FKN (grey bars) or with pCDNA 3.1 empty vector (white bars) and cultured for 24 h. PBMC were added at an effector: target ratio of 50∶1 and co-cultured with target cells for 4 h and 24 h, (n = 5). DiD positive microparticles released by target cells were enumerated by calculation of
    Figure Legend Snippet: Membrane FKN targets CD34 + -derived ECFC lysis by NK cells. ( A ) Cytotoxicity assay performed using PBMC effector cells isolated from 7 independent blood donors as described in Methods . Each independent assay was performed in triplicate (n = 7) and analysed using an effector: target ratio = 100∶1. ( B ) Representative illustration of ECFC lysis. CMFDA labeled target cells were analyzed by epi-fluorescence microscopy on an inverted microscope Nikon Eclipse TE 2000-U with a Plan Fluor 4x /0.13 objective, images were acquired using NIS elements AR software. ( C ) FKN enhances ECFC lysis by purified NK cells. CMFDA cytotoxicity assay performed using purified NK cells as effectors, effector: target ratio = 10∶1, (n = 3). ( D ) Addition of FKN neutralizing antibody reduces ECFC lysis. FKN-transfected ECFC were treated with 30 µg/ml of anti-human FKN antibody before addition of PBMC (n = 3). ( E ) Lysis of FKN expressing ECFC resulted in increased microparticles release after 24 h of incubation with PBMC.ECFC were labeled with the lipophilic DiD tracer and transfected with pCDNA3.1 vector coding for membrane FKN (grey bars) or with pCDNA 3.1 empty vector (white bars) and cultured for 24 h. PBMC were added at an effector: target ratio of 50∶1 and co-cultured with target cells for 4 h and 24 h, (n = 5). DiD positive microparticles released by target cells were enumerated by calculation of

    Techniques Used: Derivative Assay, Lysis, Cytotoxicity Assay, Isolation, Labeling, Fluorescence, Microscopy, Inverted Microscopy, Software, Purification, Transfection, Expressing, Incubation, Plasmid Preparation, Cell Culture

    FKN expression by CD34 + -derived ECFC enhances NK cell adhesion. ( A ) Flow cytometry analysis of FKN expression by ECFC 24 h after transfection with pCDNA3.1 vector encoding membrane FKN. ( B ) FKN- and control empty vector-transfected cells were incubated for 1 h with CMFDA labeled NK cells at an effector: target ratio of 1∶1. ( B, left panel ): NK cell adhesion was analyzed by epi-fluorescence microscopy on an inverted microscope Nikon TE2000-U with a Plan Fluor 4x/0.13 objective. ( B, right panel ): The number of ECFC-adherent NK cells/field was assessed using Image J 1.43 software cell counter. ( C ) Interactions between FKN transfected ECFC and NK cells were detected by confocal microscopy. ( C, left panel ) ECFC transfected with pIRES2-EGFP vector encoding membrane FKN (shown in red) were incubated for 1 h with DiD labelled purified NK cells at an effector: target ratio of 1∶1 (shown in green). ( C, right panel ): FKN-transfected ECFC were treated with 30 µg/ml of anti-human FKN antibody prior incubation with NK cells. The number of ECFC adherent NK cells was assessed by counting DiD positive NK cells/field, using Image J 1.43 software cell counter.
    Figure Legend Snippet: FKN expression by CD34 + -derived ECFC enhances NK cell adhesion. ( A ) Flow cytometry analysis of FKN expression by ECFC 24 h after transfection with pCDNA3.1 vector encoding membrane FKN. ( B ) FKN- and control empty vector-transfected cells were incubated for 1 h with CMFDA labeled NK cells at an effector: target ratio of 1∶1. ( B, left panel ): NK cell adhesion was analyzed by epi-fluorescence microscopy on an inverted microscope Nikon TE2000-U with a Plan Fluor 4x/0.13 objective. ( B, right panel ): The number of ECFC-adherent NK cells/field was assessed using Image J 1.43 software cell counter. ( C ) Interactions between FKN transfected ECFC and NK cells were detected by confocal microscopy. ( C, left panel ) ECFC transfected with pIRES2-EGFP vector encoding membrane FKN (shown in red) were incubated for 1 h with DiD labelled purified NK cells at an effector: target ratio of 1∶1 (shown in green). ( C, right panel ): FKN-transfected ECFC were treated with 30 µg/ml of anti-human FKN antibody prior incubation with NK cells. The number of ECFC adherent NK cells was assessed by counting DiD positive NK cells/field, using Image J 1.43 software cell counter.

    Techniques Used: Expressing, Derivative Assay, Flow Cytometry, Cytometry, Transfection, Plasmid Preparation, Incubation, Labeling, Fluorescence, Microscopy, Inverted Microscopy, Software, Confocal Microscopy, Purification

    8) Product Images from "SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells"

    Article Title: SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku311

    NHERF2 overexpression enhances ERα transactivation. Three cell lines, CV-1 ( A ), MCF7 ( B ) and HepG2 ( C ), were transiently transfected with empty pcDNA vector (‘Control’) or pcDNA3.1-NHERF2 (‘NHERF2’) along with ERE-TK-Luc in each case, and the effect on ERα transactivation was determined by assay of luciferase activity, as described in Materials and Methods. Assays were performed in triplicate in three independent experiments in the presence (gray bars) or absence (white bars) of E2 and the results are represented as mean ± S.E. ( D ) NHERF2 knockdown impairs ERα transactivation. MCF7 cells were transfected with NHERF2-specific siRNA or with an unrelated siRNA (control), along with ERE-TK-Luc, in the presence of E2. The effect on endogenous NHERF2 protein levels was visualized after 48 h by WB (Top panel), and the effect on ERα transactivation was determined by luciferase assay. Results, in triplicate in three independent experiments, are represented as mean ± S.E. ( E ) NHERF2 exhibits intrinsic transcriptional activity. NHERF2 transcriptional activity was assayed using the GAL4-UAS system. MCF7 cells were transfected with Gal4-DBD-NHERF2 (gray bar) or the Gal4-DBD alone (white bar), as the control, and a luciferase reporter containing a promoter with five UAS elements in tandem (5× UAS-luciferase, top panel). Luciferase activity, from three experiments assayed in triplicate, was normalized with respect to cells expressing Gal4-DBD and represented as mean ± S.E.
    Figure Legend Snippet: NHERF2 overexpression enhances ERα transactivation. Three cell lines, CV-1 ( A ), MCF7 ( B ) and HepG2 ( C ), were transiently transfected with empty pcDNA vector (‘Control’) or pcDNA3.1-NHERF2 (‘NHERF2’) along with ERE-TK-Luc in each case, and the effect on ERα transactivation was determined by assay of luciferase activity, as described in Materials and Methods. Assays were performed in triplicate in three independent experiments in the presence (gray bars) or absence (white bars) of E2 and the results are represented as mean ± S.E. ( D ) NHERF2 knockdown impairs ERα transactivation. MCF7 cells were transfected with NHERF2-specific siRNA or with an unrelated siRNA (control), along with ERE-TK-Luc, in the presence of E2. The effect on endogenous NHERF2 protein levels was visualized after 48 h by WB (Top panel), and the effect on ERα transactivation was determined by luciferase assay. Results, in triplicate in three independent experiments, are represented as mean ± S.E. ( E ) NHERF2 exhibits intrinsic transcriptional activity. NHERF2 transcriptional activity was assayed using the GAL4-UAS system. MCF7 cells were transfected with Gal4-DBD-NHERF2 (gray bar) or the Gal4-DBD alone (white bar), as the control, and a luciferase reporter containing a promoter with five UAS elements in tandem (5× UAS-luciferase, top panel). Luciferase activity, from three experiments assayed in triplicate, was normalized with respect to cells expressing Gal4-DBD and represented as mean ± S.E.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Expressing

    9) Product Images from "Comparative Measurement of Cell-Mediated Immune Responses of Swine to the M and N Proteins of Porcine Reproductive and Respiratory Syndrome Virus ▿"

    Article Title: Comparative Measurement of Cell-Mediated Immune Responses of Swine to the M and N Proteins of Porcine Reproductive and Respiratory Syndrome Virus ▿

    Journal: Clinical and Vaccine Immunology : CVI

    doi: 10.1128/CVI.00365-09

    Antibody levels in the immunized pigs. Pigs were immunized with plasmid DNAs, and the levels antibodies to the PRRSV-M and PRRSV-N proteins were determined by ELISA. (A) Levels of antibody to the PRRSV-M protein in pigs immunized with pcDNA3.1-GM-CSF-PRRSV-M.
    Figure Legend Snippet: Antibody levels in the immunized pigs. Pigs were immunized with plasmid DNAs, and the levels antibodies to the PRRSV-M and PRRSV-N proteins were determined by ELISA. (A) Levels of antibody to the PRRSV-M protein in pigs immunized with pcDNA3.1-GM-CSF-PRRSV-M.

    Techniques Used: Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    10) Product Images from "Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-?B and proinflammatory gene program activation in intestinal epithelial cells"

    Article Title: Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-?B and proinflammatory gene program activation in intestinal epithelial cells

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-4-33

    TLR5 inhibits flagellin-mediated NF-κB reporter gene activity. HT29 cells were transfected in triplicate in 6-well dishes using the indicated DN-TLR mammalian expression vectors or antisense TLR5 (AS TLR5) (2 μg/well), 2× NF-κB Luc reporter gene (100 ng/well), pRL-TK Renilla luciferase for normalization (50 ng/well) adjusted to 4 μg total DNA/well with empty vector pCDNA3.1 DNA. A, Fold-induction of 2× NF-κB Luc reporter gene in non-stimulated cells (light shading) and in TNFα (10 ng/ml) treated cells (dark shading). Lysates were prepared 12 h after stimulation. Results of a representative experiment are shown. B, HT29 cells transfected as in A were treated with flagellin (1 μg/ml) and cell lysates were prepared and analyzed as in Fig. 8A. Results of a representative experiment are shown.
    Figure Legend Snippet: TLR5 inhibits flagellin-mediated NF-κB reporter gene activity. HT29 cells were transfected in triplicate in 6-well dishes using the indicated DN-TLR mammalian expression vectors or antisense TLR5 (AS TLR5) (2 μg/well), 2× NF-κB Luc reporter gene (100 ng/well), pRL-TK Renilla luciferase for normalization (50 ng/well) adjusted to 4 μg total DNA/well with empty vector pCDNA3.1 DNA. A, Fold-induction of 2× NF-κB Luc reporter gene in non-stimulated cells (light shading) and in TNFα (10 ng/ml) treated cells (dark shading). Lysates were prepared 12 h after stimulation. Results of a representative experiment are shown. B, HT29 cells transfected as in A were treated with flagellin (1 μg/ml) and cell lysates were prepared and analyzed as in Fig. 8A. Results of a representative experiment are shown.

    Techniques Used: Activity Assay, Transfection, Expressing, Luciferase, Plasmid Preparation

    11) Product Images from "Inorganic polyphosphate stimulates mammalian TOR, a kinase involved in the proliferation of mammary cancer cells"

    Article Title: Inorganic polyphosphate stimulates mammalian TOR, a kinase involved in the proliferation of mammary cancer cells

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1534805100

    Polyphosphatase PPX1 reduces insulin and amino acid activation of mTOR activity in vivo . Serum- and amino acid-starved MCF-7 cells, either the pcDNA3.1 transfected (pcDNA#1) or PPX1-expressing (PPX1#1) cells, were incubated for 30 min with either 0.1 or 0.5 of the normal levels of amino acids in MEM or 10 nM or 100 nM insulin, as indicated. Cell lysates were analyzed by immunoblotting for an extraneous protein (IDE), total PHAS-I levels, or PHAS-I that was phosphorylated on Ser-65. ( Upper ) Western blots of a representative experiment with arrows indicating the positions of IDE, total PHAS-I protein, and phosphorylated PHAS-I. The amount of pPHAS-I was quantitated, and shown are the averages of three experiments ± SE expressed as a percent of the maximally stimulated cells.
    Figure Legend Snippet: Polyphosphatase PPX1 reduces insulin and amino acid activation of mTOR activity in vivo . Serum- and amino acid-starved MCF-7 cells, either the pcDNA3.1 transfected (pcDNA#1) or PPX1-expressing (PPX1#1) cells, were incubated for 30 min with either 0.1 or 0.5 of the normal levels of amino acids in MEM or 10 nM or 100 nM insulin, as indicated. Cell lysates were analyzed by immunoblotting for an extraneous protein (IDE), total PHAS-I levels, or PHAS-I that was phosphorylated on Ser-65. ( Upper ) Western blots of a representative experiment with arrows indicating the positions of IDE, total PHAS-I protein, and phosphorylated PHAS-I. The amount of pPHAS-I was quantitated, and shown are the averages of three experiments ± SE expressed as a percent of the maximally stimulated cells.

    Techniques Used: Activation Assay, Activity Assay, In Vivo, Transfection, Expressing, Incubation, Western Blot

    12) Product Images from "A Novel Family of Adhesion-Like Molecules That Interacts with the NMDA Receptor"

    Article Title: A Novel Family of Adhesion-Like Molecules That Interacts with the NMDA Receptor

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3799-05.2006

    SALM1 promotes neurite outgrowth in cultured hippocampal neurons. Cultured hippocampal neurons at 4 DIV were cotransfected with GFP plus pcDNA3.1 vector ( a ), GFP plus SALM1 ( b ), or GFP plus SALM1-ΔCT ( c ) and stained with anti-SALM1 and anti-GFP antibodies. Cells were processed 48 h after transfection. The total neurite length of each neuron was traced based on GFP distribution and measured using MetaMorph software. The total neurite length per cell is 1795 ± 649 μm for GFP plus pcDNA3.1 vector, 3807 ± 1219 μm for GFP plus SALM1, and 1558 ± 403 μm for GFP plus SALM1-ΔCT ( g ), obtained from 42, 54, and 10 neurons, respectively. Hippocampal neurons at 14 DIV were also transfected with GFP plus SALM1-myc and GFP plus SALM1-ΔCTmyc ( d–f , h ). Overexpression of SALM1-ΔCTmyc had a dramatic effect on dendritic morphology of these neurons ( f ) with the appearance of more filopodia-like structures. The number of protrusions per 10 μm for GFP plus pcDNA3.1 is 3.7 ± 1.1, for GFP plus SALM1-myc is 3.2 ± 0.7, and for GFP plus SALM1-ΔCTmyc is 7.4 ± 1.5 ( h ). Data were obtained from 10 neurons for each condition. Scale bars: a–c , d–f , top panels, 20 μm; e , f , bottom panels, 10 μm. * p
    Figure Legend Snippet: SALM1 promotes neurite outgrowth in cultured hippocampal neurons. Cultured hippocampal neurons at 4 DIV were cotransfected with GFP plus pcDNA3.1 vector ( a ), GFP plus SALM1 ( b ), or GFP plus SALM1-ΔCT ( c ) and stained with anti-SALM1 and anti-GFP antibodies. Cells were processed 48 h after transfection. The total neurite length of each neuron was traced based on GFP distribution and measured using MetaMorph software. The total neurite length per cell is 1795 ± 649 μm for GFP plus pcDNA3.1 vector, 3807 ± 1219 μm for GFP plus SALM1, and 1558 ± 403 μm for GFP plus SALM1-ΔCT ( g ), obtained from 42, 54, and 10 neurons, respectively. Hippocampal neurons at 14 DIV were also transfected with GFP plus SALM1-myc and GFP plus SALM1-ΔCTmyc ( d–f , h ). Overexpression of SALM1-ΔCTmyc had a dramatic effect on dendritic morphology of these neurons ( f ) with the appearance of more filopodia-like structures. The number of protrusions per 10 μm for GFP plus pcDNA3.1 is 3.7 ± 1.1, for GFP plus SALM1-myc is 3.2 ± 0.7, and for GFP plus SALM1-ΔCTmyc is 7.4 ± 1.5 ( h ). Data were obtained from 10 neurons for each condition. Scale bars: a–c , d–f , top panels, 20 μm; e , f , bottom panels, 10 μm. * p

    Techniques Used: Cell Culture, Plasmid Preparation, Staining, Transfection, Software, Over Expression

    13) Product Images from "Lipoteichoic Acids from Lactobacillus Strains Elicit Strong Tumor Necrosis Factor Alpha-Inducing Activities in Macrophages through Toll-Like Receptor 2"

    Article Title: Lipoteichoic Acids from Lactobacillus Strains Elicit Strong Tumor Necrosis Factor Alpha-Inducing Activities in Macrophages through Toll-Like Receptor 2

    Journal: Clinical and Diagnostic Laboratory Immunology

    doi: 10.1128/CDLI.10.2.259-266.2003

    Signal transduction induced by Lactobacillus spp. (A) HEK293T cells were transiently transfected with 0.5 μg of each of pcDNA3.1(+)-mCD14, pGL3-NF-kB-luc, and pRL-SV40 (as an internal control), plus 0.5 μg of one of the following: pcDNA3.1(+) (EV), pEFBOS-mTLR2/Flag (TLR2), or p3XFlag-mTLR4 (TLR4). At 48 h after the transfection, the cells were untreated, treated with LTA (1 μg/ml) of L. fermentum (F) or L. casei (C) or with synthetic lipid A (1 μg/ml) (A) for 8 h, and then lysed to measure the luciferase activities. The experiment was repeated three times, yielding similar results, and a typical result is shown. (B) HEK293T cells were transiently transfected with 0.5 μg each of pcDNA3.1(+)-mCD14, pGL3-NF-kB-luc, and pRL-SV40, plus 1 μg of one of the following: pcDNA3.1(+) (EV), pEFBOS-mTLR2/Flag (TLR2) plus pcDNA3/1(+) (0.5 μg each), or the combination of pEFBOAS-mTLR2/Flag and pEFBOS-mTLR6/Flag (0.5 μg/each) (TLR2 + TLR6). At 48 h after the transfection, the cells were either not treated or treated with heat-killed L. fermentum (1 μg/ml) for 8 h and then lysed for the measurement of the luciferase activities. The experiment was repeated three times, yielding similar results, and a typical result is shown. (C) Lactobacillus LTA induces JNK activation in RAW264.7 cells. RAW264.7 cells were either not treated or treated with 1 μg of either LPS or purified LTA from L. fermentum /ml for 30 min. Cells were lysed, and the JNK1 kinase activity was measured by using GST-c-Jun as the substrate.
    Figure Legend Snippet: Signal transduction induced by Lactobacillus spp. (A) HEK293T cells were transiently transfected with 0.5 μg of each of pcDNA3.1(+)-mCD14, pGL3-NF-kB-luc, and pRL-SV40 (as an internal control), plus 0.5 μg of one of the following: pcDNA3.1(+) (EV), pEFBOS-mTLR2/Flag (TLR2), or p3XFlag-mTLR4 (TLR4). At 48 h after the transfection, the cells were untreated, treated with LTA (1 μg/ml) of L. fermentum (F) or L. casei (C) or with synthetic lipid A (1 μg/ml) (A) for 8 h, and then lysed to measure the luciferase activities. The experiment was repeated three times, yielding similar results, and a typical result is shown. (B) HEK293T cells were transiently transfected with 0.5 μg each of pcDNA3.1(+)-mCD14, pGL3-NF-kB-luc, and pRL-SV40, plus 1 μg of one of the following: pcDNA3.1(+) (EV), pEFBOS-mTLR2/Flag (TLR2) plus pcDNA3/1(+) (0.5 μg each), or the combination of pEFBOAS-mTLR2/Flag and pEFBOS-mTLR6/Flag (0.5 μg/each) (TLR2 + TLR6). At 48 h after the transfection, the cells were either not treated or treated with heat-killed L. fermentum (1 μg/ml) for 8 h and then lysed for the measurement of the luciferase activities. The experiment was repeated three times, yielding similar results, and a typical result is shown. (C) Lactobacillus LTA induces JNK activation in RAW264.7 cells. RAW264.7 cells were either not treated or treated with 1 μg of either LPS or purified LTA from L. fermentum /ml for 30 min. Cells were lysed, and the JNK1 kinase activity was measured by using GST-c-Jun as the substrate.

    Techniques Used: Transduction, Transfection, Luciferase, Activation Assay, Purification, Activity Assay

    14) Product Images from "Non-structural protein 1 of avian influenza A viruses differentially inhibit NF-?B promoter activation"

    Article Title: Non-structural protein 1 of avian influenza A viruses differentially inhibit NF-?B promoter activation

    Journal: Virology Journal

    doi: 10.1186/1743-422X-8-383

    Expression patterns of allele A and allele B NS1 proteins in subcellular compartments in A549 and MiLu cells . (A) A549 cells were transfected with vector encoding NS1 from H6N8-A, H6N8-B, H4N6-A, H4N6-B or empty pcDNA3.1+ vector (mock treated). 24 hours post-transfection cells were lysed and subjected to Western blotting and probed with anti-NS1 and β-actin antibodies. (B) A549 and MiLu cells were transfected with vector encoding NS1 from H6N8-A, H6N8-B, H4N6-A, H4N6-B. 18 hours post transfection, cells were fixed and processed for in situ PLA, as recommended.
    Figure Legend Snippet: Expression patterns of allele A and allele B NS1 proteins in subcellular compartments in A549 and MiLu cells . (A) A549 cells were transfected with vector encoding NS1 from H6N8-A, H6N8-B, H4N6-A, H4N6-B or empty pcDNA3.1+ vector (mock treated). 24 hours post-transfection cells were lysed and subjected to Western blotting and probed with anti-NS1 and β-actin antibodies. (B) A549 and MiLu cells were transfected with vector encoding NS1 from H6N8-A, H6N8-B, H4N6-A, H4N6-B. 18 hours post transfection, cells were fixed and processed for in situ PLA, as recommended.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, In Situ, Proximity Ligation Assay

    Allele A and B NS1 proteins of avian influenza A viruses differentially block the dsRNA-induced expression of NF-κB promoter in A549 and MiLu cells . (A) A549 cells were transiently co-transfected with NF-κB luciferase reporter plasmid, and vector encoding NS1 from H6N8-A, H6N8-B, H4N6-A, H4N6-B or empty pcDNA3.1+ vector (mock treated) or left un-transfected (cells). 24 hours post transfection, cells were stimulated with dsRNA (+) or left un-stimulated (-). 24 hours post induction, cell extracts were prepared and luciferase activity was measured using the ONE-Glo™ Luciferase Assay System (Promega) following the manufacturer's Instructions. The values were normalized and pNF-κB cont. was set to 100%. (B) MiLu cells were transfected as described for A549 cells. All the conditions were maintained for MiLu cells as that of (A). Error bars indicate standard deviations. The data shown are representative for three experiments with transfections performed in duplicate. * indicates a significant difference as determined by the student's t-test, with p-values of
    Figure Legend Snippet: Allele A and B NS1 proteins of avian influenza A viruses differentially block the dsRNA-induced expression of NF-κB promoter in A549 and MiLu cells . (A) A549 cells were transiently co-transfected with NF-κB luciferase reporter plasmid, and vector encoding NS1 from H6N8-A, H6N8-B, H4N6-A, H4N6-B or empty pcDNA3.1+ vector (mock treated) or left un-transfected (cells). 24 hours post transfection, cells were stimulated with dsRNA (+) or left un-stimulated (-). 24 hours post induction, cell extracts were prepared and luciferase activity was measured using the ONE-Glo™ Luciferase Assay System (Promega) following the manufacturer's Instructions. The values were normalized and pNF-κB cont. was set to 100%. (B) MiLu cells were transfected as described for A549 cells. All the conditions were maintained for MiLu cells as that of (A). Error bars indicate standard deviations. The data shown are representative for three experiments with transfections performed in duplicate. * indicates a significant difference as determined by the student's t-test, with p-values of

    Techniques Used: Blocking Assay, Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay

    Impact of RNA binding domain and effector domain of NS1 protein on dsRNA induced NF-κB promoter activity in A549 and MiLu cells . (A) A schematic presentation for the construction of the RNA binding domain, the effector domain from both alleles of H6N8 and chimeric NS1s. (B) A549 cells were transiently co-transfected with NF-κB luciferase reporter plasmid, and vector encoding NS1 from H6N8-A, H6N8-B, H6N8-A-RNA, H6N8-A-ED, H6N8-B-RNA, H6N8-B-ED, H6N8 chiNS1 A/B, H6N8 chiNS1 B/A or empty pcDNA3.1+ vector (pNF-κB cont.) or left un-transfected (cells). 24 hours post transfection, cells were stimulated with dsRNA, cell extracts were prepared and luciferase activities were measured using the ONE-Glo™ Luciferase Assay System (Promega). The values were normalized and pNF-κB cont. was set to 100%. (C) All the conditions in MiLu cells transfection were maintained as practiced in (B). Error bars indicate standard deviations. The data shown are representative for three experiments with transfections performed in duplicate. * indicates a significant difference as determined by the student's t-test, with p-values of
    Figure Legend Snippet: Impact of RNA binding domain and effector domain of NS1 protein on dsRNA induced NF-κB promoter activity in A549 and MiLu cells . (A) A schematic presentation for the construction of the RNA binding domain, the effector domain from both alleles of H6N8 and chimeric NS1s. (B) A549 cells were transiently co-transfected with NF-κB luciferase reporter plasmid, and vector encoding NS1 from H6N8-A, H6N8-B, H6N8-A-RNA, H6N8-A-ED, H6N8-B-RNA, H6N8-B-ED, H6N8 chiNS1 A/B, H6N8 chiNS1 B/A or empty pcDNA3.1+ vector (pNF-κB cont.) or left un-transfected (cells). 24 hours post transfection, cells were stimulated with dsRNA, cell extracts were prepared and luciferase activities were measured using the ONE-Glo™ Luciferase Assay System (Promega). The values were normalized and pNF-κB cont. was set to 100%. (C) All the conditions in MiLu cells transfection were maintained as practiced in (B). Error bars indicate standard deviations. The data shown are representative for three experiments with transfections performed in duplicate. * indicates a significant difference as determined by the student's t-test, with p-values of

    Techniques Used: RNA Binding Assay, Activity Assay, Transfection, Luciferase, Plasmid Preparation

    15) Product Images from "FoxM1 Promotes Glioma Cells Progression by Up-Regulating Anxa1 Expression"

    Article Title: FoxM1 Promotes Glioma Cells Progression by Up-Regulating Anxa1 Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072376

    Effect of FoxM1/Anxa1 expression on proliferation and migration of glioma cells in vitro . A, RT-qPCR and Western blot analyses of FoxM1 and Anxa1 expression in stable pcDNA3.1-Anxa1-transfected U-87MG-RNAi cells (left) and Anxa1-shRNA-transfected SW0188-FoxM1 cells (right). B, Cells as in (A) were cultured in 96-well plates and analyzed by MTT assay. Cell proliferation curves were shown in 9 days. Three independent experiments were conducted. C, Cells as in (A) were examined for cell migration motility in 24-well plates with transwell chambers. Migrated cells were stained with crystal violet (upper) and counted under a light microscope (lower). Three independent experiments were conducted. D, the angiogenic potential of glioma cells was determined by endothelial cell tube formation assay. Capillary tube formation in each group was photographed and quantified. * P
    Figure Legend Snippet: Effect of FoxM1/Anxa1 expression on proliferation and migration of glioma cells in vitro . A, RT-qPCR and Western blot analyses of FoxM1 and Anxa1 expression in stable pcDNA3.1-Anxa1-transfected U-87MG-RNAi cells (left) and Anxa1-shRNA-transfected SW0188-FoxM1 cells (right). B, Cells as in (A) were cultured in 96-well plates and analyzed by MTT assay. Cell proliferation curves were shown in 9 days. Three independent experiments were conducted. C, Cells as in (A) were examined for cell migration motility in 24-well plates with transwell chambers. Migrated cells were stained with crystal violet (upper) and counted under a light microscope (lower). Three independent experiments were conducted. D, the angiogenic potential of glioma cells was determined by endothelial cell tube formation assay. Capillary tube formation in each group was photographed and quantified. * P

    Techniques Used: Expressing, Migration, In Vitro, Quantitative RT-PCR, Western Blot, Transfection, shRNA, Cell Culture, MTT Assay, Staining, Light Microscopy, Endothelial Cell Tube Formation Assay

    16) Product Images from "Thioredoxin Confers Intrinsic Resistance to Cytostatic Drugs in Human Glioma Cells"

    Article Title: Thioredoxin Confers Intrinsic Resistance to Cytostatic Drugs in Human Glioma Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19102874

    Overexpression of the tumour suppressor TXNIP inhibits Trx expression and sensitizes GBM cell lines to cisplatin. ( a ) Representative Western blots of U87 cells showing overexpression of TXNIP and downregulation of Trx-1 protein expression after transient transfection with pcDNA3.1-TXNIP or empty vector (pcDNA3.1) 48 h post-transfection. β-actin Western blot was performed to control for loading; ( b – d ) MTT assays with cisplatin concentration-response curves showing sensitization of U87 ( b ), U251 ( c ), and U373 ( d ) cells after transient transfection with empty vector (pcDNA3.1) or pcDNA3.1-TXNIP. Cells were incubated with cisplatin for 72 h at 24 h post-transfection; ( e ) Respective cisplatin IC 50 values derived from curves presented in b – d ( n = 4, * p
    Figure Legend Snippet: Overexpression of the tumour suppressor TXNIP inhibits Trx expression and sensitizes GBM cell lines to cisplatin. ( a ) Representative Western blots of U87 cells showing overexpression of TXNIP and downregulation of Trx-1 protein expression after transient transfection with pcDNA3.1-TXNIP or empty vector (pcDNA3.1) 48 h post-transfection. β-actin Western blot was performed to control for loading; ( b – d ) MTT assays with cisplatin concentration-response curves showing sensitization of U87 ( b ), U251 ( c ), and U373 ( d ) cells after transient transfection with empty vector (pcDNA3.1) or pcDNA3.1-TXNIP. Cells were incubated with cisplatin for 72 h at 24 h post-transfection; ( e ) Respective cisplatin IC 50 values derived from curves presented in b – d ( n = 4, * p

    Techniques Used: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, MTT Assay, Concentration Assay, Incubation, Derivative Assay

    17) Product Images from "Heterodimerization of Glycosylated Insulin-Like Growth Factor-1 Receptors and Insulin Receptors in Cancer Cells Sensitive to Anti-IGF1R Antibody"

    Article Title: Heterodimerization of Glycosylated Insulin-Like Growth Factor-1 Receptors and Insulin Receptors in Cancer Cells Sensitive to Anti-IGF1R Antibody

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033322

    Identification of a specific N-linked glycosylation site (N913) of IGF1R in sensitive cell lines and its functional importance in the response to figitumumab. A) Identification of the NLG site occupancy of IGF1Rβ subunits. IGF1Rβ subunits containing N-linked glycosylation sites (Asn747, Asn756, Asn764, Asn900, and Asn913) were isolated from both drug sensitive (SNU719, HepG2 and SNU368) and resistance (SNU638 and SNU354) cells and identified by tandem MS by an increase of 1.0 Da from the corresponding mass of Asn as a result of conversion from N-linked glycosylated Asn to Asp. All the NLG at Asn900 in both sensitive and resistance cells were determined to be occupied with N-glycosylation (filled rectangle). NLG at Asn913 of the sensitive cell lines (HepG2, SNU719, and SNU368) were determined to be occupied with N-glycosylation (filled rectangle), whereas N-glycosites at Asn913 of the resistance cell lines (SNU638 and SNU354) were found to be unoccupied with N-glycosylation. (open rectangle). B) Effect of the N913Q site mutation on electrophoretic mobility patterns of IGF1Rβ. Huh7 cells (an IGF1R-negative cell line) were transfected with the empty pcDNA3.1(-) expression vector(Control), pcDNA3.1(-) containing wild-type IGF1R cDNA (IGF1R WT), or pcDNA3.1(-) with IGF1R mutation type cDNA (IGF1R N913Q). An equal amount of the cell lysate from the transfected cells was then subjected to Western blot analysis for IGF1Rβ. C) Effect of N913Q site mutation on the formation of IGF1R/IR heterodimeric receptors. An equal amount of the cell lysate from transfected cells was then subjected to immunoprecipitation (IP) with anti-IGF1R antibody followed by Western blot analysis for IRβ and IGF1Rβ. Input = total cell lysate without IP. D) Effect of N913Q site mutation on IGF1R localization. An immunofluoresence assay was conducted to observe the localization of IGF1R. IGF1R reactivity was visualized by confocal laser scanning microscopy (Scale bar: 30 µm). Representative images are shown. Green: IGF1R, Blue: nuclei. E) Effect of N913Q site mutation on figitumumab sensitivity. Huh7 cells transfected with empty pcDNA3.1(-) vector, vector containing wild-type IGF1R cDNA, or vector containing IGF1R (N913Q) mutation type cDNA were plated in 96-well plates and treated with increasing concentrations of figitumumab for 120 h (left). Cell viability percentages with 1 µg/mL figitumumab (right). Six replicate wells were included in each analysis, and at least three independent experiments were conducted. Data from replicate wells are presented as the mean of remaining cells. * P- values
    Figure Legend Snippet: Identification of a specific N-linked glycosylation site (N913) of IGF1R in sensitive cell lines and its functional importance in the response to figitumumab. A) Identification of the NLG site occupancy of IGF1Rβ subunits. IGF1Rβ subunits containing N-linked glycosylation sites (Asn747, Asn756, Asn764, Asn900, and Asn913) were isolated from both drug sensitive (SNU719, HepG2 and SNU368) and resistance (SNU638 and SNU354) cells and identified by tandem MS by an increase of 1.0 Da from the corresponding mass of Asn as a result of conversion from N-linked glycosylated Asn to Asp. All the NLG at Asn900 in both sensitive and resistance cells were determined to be occupied with N-glycosylation (filled rectangle). NLG at Asn913 of the sensitive cell lines (HepG2, SNU719, and SNU368) were determined to be occupied with N-glycosylation (filled rectangle), whereas N-glycosites at Asn913 of the resistance cell lines (SNU638 and SNU354) were found to be unoccupied with N-glycosylation. (open rectangle). B) Effect of the N913Q site mutation on electrophoretic mobility patterns of IGF1Rβ. Huh7 cells (an IGF1R-negative cell line) were transfected with the empty pcDNA3.1(-) expression vector(Control), pcDNA3.1(-) containing wild-type IGF1R cDNA (IGF1R WT), or pcDNA3.1(-) with IGF1R mutation type cDNA (IGF1R N913Q). An equal amount of the cell lysate from the transfected cells was then subjected to Western blot analysis for IGF1Rβ. C) Effect of N913Q site mutation on the formation of IGF1R/IR heterodimeric receptors. An equal amount of the cell lysate from transfected cells was then subjected to immunoprecipitation (IP) with anti-IGF1R antibody followed by Western blot analysis for IRβ and IGF1Rβ. Input = total cell lysate without IP. D) Effect of N913Q site mutation on IGF1R localization. An immunofluoresence assay was conducted to observe the localization of IGF1R. IGF1R reactivity was visualized by confocal laser scanning microscopy (Scale bar: 30 µm). Representative images are shown. Green: IGF1R, Blue: nuclei. E) Effect of N913Q site mutation on figitumumab sensitivity. Huh7 cells transfected with empty pcDNA3.1(-) vector, vector containing wild-type IGF1R cDNA, or vector containing IGF1R (N913Q) mutation type cDNA were plated in 96-well plates and treated with increasing concentrations of figitumumab for 120 h (left). Cell viability percentages with 1 µg/mL figitumumab (right). Six replicate wells were included in each analysis, and at least three independent experiments were conducted. Data from replicate wells are presented as the mean of remaining cells. * P- values

    Techniques Used: Functional Assay, Isolation, Mass Spectrometry, Mutagenesis, Transfection, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation, Confocal Laser Scanning Microscopy

    Effect of selective IR overexpression on HR levels and the anti-proliferative effect of figitumumab in IR-transfected cells. A) Effect of IR transfection on IGF1R/IR HR levels in IR-negative cell lines. Cells were transfected with the pcDNA3.1(-) expression vector containing wild-type IR cDNA. An equal amount of lysates from cells transfected with either the empty vector or pcDNA3.1(-) containing IR cDNA was subjected to immunoprecipitation with an anti-IGF1R antibody followed by Western blot analyses of IRβ and IGF1Rβ. B) Effect of IR-transfection on figitumumab sensitivity. Transfected cells were plated onto 96-well plates, treated with figitumumab for 5 d, and subjected to MTT assays. Solid triangle symbol with dashed lines = empty vector (pcDNA3.1-), Solid circle symbols with lines = pcDNA3.1(-) IR. Bar = ±SE. Mean values were derived from six replicates. Experiments were repeated in triplicate.
    Figure Legend Snippet: Effect of selective IR overexpression on HR levels and the anti-proliferative effect of figitumumab in IR-transfected cells. A) Effect of IR transfection on IGF1R/IR HR levels in IR-negative cell lines. Cells were transfected with the pcDNA3.1(-) expression vector containing wild-type IR cDNA. An equal amount of lysates from cells transfected with either the empty vector or pcDNA3.1(-) containing IR cDNA was subjected to immunoprecipitation with an anti-IGF1R antibody followed by Western blot analyses of IRβ and IGF1Rβ. B) Effect of IR-transfection on figitumumab sensitivity. Transfected cells were plated onto 96-well plates, treated with figitumumab for 5 d, and subjected to MTT assays. Solid triangle symbol with dashed lines = empty vector (pcDNA3.1-), Solid circle symbols with lines = pcDNA3.1(-) IR. Bar = ±SE. Mean values were derived from six replicates. Experiments were repeated in triplicate.

    Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, MTT Assay, Derivative Assay

    18) Product Images from "KIF5B gene sequence variation and response of cardiac stroke volume to regular exercise "

    Article Title: KIF5B gene sequence variation and response of cardiac stroke volume to regular exercise

    Journal: Physiological Genomics

    doi: 10.1152/physiolgenomics.00003.2008

    Effects of kif5b overexpression on mitochondrial content of C2C12 cells. A : immunostaining of cells transfected with the negative control empty vector pcDNA3.1 had no effect on kif5b expression and mitochondrial content. Transfected cells are shown in bright green by GFP. B : Kif5b expression and mitochondrial staining were significantly increased in cells transfected with the kif5b expression construct, identified by the GFP. C : overexpression of kif5b led to significant increase of kif5b mRNA and protein levels ( D ). E : transient transfection of cells with the kif5b expression construct increased mitochondrial biogenesis as measured by the increase of mitochondrial cytochrome b adjusted by nuclear DNA. * P
    Figure Legend Snippet: Effects of kif5b overexpression on mitochondrial content of C2C12 cells. A : immunostaining of cells transfected with the negative control empty vector pcDNA3.1 had no effect on kif5b expression and mitochondrial content. Transfected cells are shown in bright green by GFP. B : Kif5b expression and mitochondrial staining were significantly increased in cells transfected with the kif5b expression construct, identified by the GFP. C : overexpression of kif5b led to significant increase of kif5b mRNA and protein levels ( D ). E : transient transfection of cells with the kif5b expression construct increased mitochondrial biogenesis as measured by the increase of mitochondrial cytochrome b adjusted by nuclear DNA. * P

    Techniques Used: Over Expression, Immunostaining, Transfection, Negative Control, Plasmid Preparation, Expressing, Staining, Construct

    19) Product Images from "TonEBP/NFAT5 Stimulates Transcription of HSP70 in Response to Hypertonicity"

    Article Title: TonEBP/NFAT5 Stimulates Transcription of HSP70 in Response to Hypertonicity

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.16.5753-5760.2002

    Activity of HSP70-2 promoter in response to hypertonicity and heat shock. A 4.1-kb genomic DNA fragment covering nucleotides −4,158 to −43 upstream of the start codon was cloned into a luciferase reporter vector. mIMCD cells were transfected with the reporter plasmid along with pcDNA3.1 (vector) or pcDNA3.1 driving expression of DN-TonEBP or TonEBP as indicated. The transfected cells were treated with hypertonic medium or heat shock. The activity of luciferase is shown relative to the vector control. Data are the mean ± standard deviation ( n = 4). Note that the scale for the heat shock-treated group is shown on the right.
    Figure Legend Snippet: Activity of HSP70-2 promoter in response to hypertonicity and heat shock. A 4.1-kb genomic DNA fragment covering nucleotides −4,158 to −43 upstream of the start codon was cloned into a luciferase reporter vector. mIMCD cells were transfected with the reporter plasmid along with pcDNA3.1 (vector) or pcDNA3.1 driving expression of DN-TonEBP or TonEBP as indicated. The transfected cells were treated with hypertonic medium or heat shock. The activity of luciferase is shown relative to the vector control. Data are the mean ± standard deviation ( n = 4). Note that the scale for the heat shock-treated group is shown on the right.

    Techniques Used: Activity Assay, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Expressing, Standard Deviation

    Effects of DN-TonEBP on mRNA expression. MDCK cells stably transfected with pcDNA3.1 (lanes V) or pcDNA3.1 driving expression of DN-TonEBP (lanes R6, a clonal line) were cultured overnight in control isotonic (lanes C) or hypertonic medium (lanes HT). (A) Cell lysate containing 40 μg of protein was used in each lane for immunoblot analysis of TonEBP. Bands representing TonEBP and DN-TonEBP are indicated on the right. (B) Five micrograms of RNA was used for Northern blot detection of mRNA for SMIT, BGT1, AR, HSP70, COX-2, αB-crystallin, and glyceraldehyde 3-phosphate dehydrogenase.
    Figure Legend Snippet: Effects of DN-TonEBP on mRNA expression. MDCK cells stably transfected with pcDNA3.1 (lanes V) or pcDNA3.1 driving expression of DN-TonEBP (lanes R6, a clonal line) were cultured overnight in control isotonic (lanes C) or hypertonic medium (lanes HT). (A) Cell lysate containing 40 μg of protein was used in each lane for immunoblot analysis of TonEBP. Bands representing TonEBP and DN-TonEBP are indicated on the right. (B) Five micrograms of RNA was used for Northern blot detection of mRNA for SMIT, BGT1, AR, HSP70, COX-2, αB-crystallin, and glyceraldehyde 3-phosphate dehydrogenase.

    Techniques Used: Expressing, Stable Transfection, Transfection, Cell Culture, Northern Blot

    20) Product Images from "DC targeting DNA vaccines induce protective and therapeutic antitumor immunity in mice"

    Article Title: DC targeting DNA vaccines induce protective and therapeutic antitumor immunity in mice

    Journal: International Journal of Clinical and Experimental Medicine

    doi:

    Plasmid DNA vaccines encoding secreted scFv N418 -HER2/neu fusion proteins. A. Schematic representation of expression vectors. scFv N418 -HER2, scFv N418 -neu, pcDNA3.1-HER2, or pcDNA3.1-neu encode under the control of a CMV promoter, all the fusion proteins consisting of an signal peptide, amino acid residues 1 to 222 of human HER2 or amino acid residues 1 to 224 of rat neu, and COOH-terminal Myc tag. The control plasmids pcDNA3.1-HER2 and pcDNA3.1-neu lack the scFv N418 domain. B. 293T cells grown in 100-mm dishes were transfected with various expression vectors using Lipofectamine 2000 (invitrogen). Immunoblot analysis of supernatants from 293T cells transfected with scFv N418 -HER2, scFv N418 -neu, pcDNA3.1-HER2 and pcDNA3.1-neu (lane 1, 2, 3 and 4). Vaccine proteins were probed with mouse anti-Myc tag mAb followed by HRP-conjugated secondary anti-mouse antibody. C. Binding of scFv N418 -HER2 fusion protein from culture supernatant of transfected 293T cells to mouse dendrtic cells was investigated by fluorescence-activated cell sorting analysis with mAb 9E10 followed by PE-conjugated secondary antibody. Control cells were treated with supernatant from mock-transfected cells.
    Figure Legend Snippet: Plasmid DNA vaccines encoding secreted scFv N418 -HER2/neu fusion proteins. A. Schematic representation of expression vectors. scFv N418 -HER2, scFv N418 -neu, pcDNA3.1-HER2, or pcDNA3.1-neu encode under the control of a CMV promoter, all the fusion proteins consisting of an signal peptide, amino acid residues 1 to 222 of human HER2 or amino acid residues 1 to 224 of rat neu, and COOH-terminal Myc tag. The control plasmids pcDNA3.1-HER2 and pcDNA3.1-neu lack the scFv N418 domain. B. 293T cells grown in 100-mm dishes were transfected with various expression vectors using Lipofectamine 2000 (invitrogen). Immunoblot analysis of supernatants from 293T cells transfected with scFv N418 -HER2, scFv N418 -neu, pcDNA3.1-HER2 and pcDNA3.1-neu (lane 1, 2, 3 and 4). Vaccine proteins were probed with mouse anti-Myc tag mAb followed by HRP-conjugated secondary anti-mouse antibody. C. Binding of scFv N418 -HER2 fusion protein from culture supernatant of transfected 293T cells to mouse dendrtic cells was investigated by fluorescence-activated cell sorting analysis with mAb 9E10 followed by PE-conjugated secondary antibody. Control cells were treated with supernatant from mock-transfected cells.

    Techniques Used: Plasmid Preparation, Expressing, Transfection, Binding Assay, Fluorescence, FACS

    Vaccination with scFv N418 -HER2 induced HER2-specific cellular and antibody immune response. BALB/c mice were vaccinated with HER2, scFv N418 -neu or scFv N418 -HER2. Control animals received pcDNA3.1. The draining lymph nodes and spleens were harvested from the vaccinated animals after two vaccinations. A. CD4 + T cells isolated from the draining lymph were cultured in the presence of 10 µg/ml recombinant HER2 or TRP2 protein for 4 d with the addition of [ 3 H] thymidine in the last 16 h. T-cell proliferation was determined by [ 3 H] thymidine incorporation (left panel). Right panel, the supernatant recovered from the assay in left was tested for cytokine production by ELISA. B. Splenocytes isolated from the vaccinated animals were stimulated for 6 h with H-2K d -restricted HER2 63-71 peptide TYLPTNASL before flow cytometric analysis with anti-CD8 and anti-IFN-γ antibodies. Left, increase in CD8 + IFN-γ + cells upon peptide stimulation of splenocytes. Representative results from one animal of scFv N418 -HER2 group upon restimulation in the presence of HER2 or TRP2 peptide. Right, absolute numbers of CD8 + IFN-γ + splenocytes (mean values from five mice per group). C. Splenocytes were cocultured with D2F2/E2 cells for 5 d. The resultant splenocytes (E) were cocultured for 4 h with the 51 Cr-labeled target cells (T). Percentages of target cells killing by the splenocytes from the vaccinated mice are shown. Data represent the means of triplicate cultures and are representative of two independent experiments. D. HER2-specific total IgG and IgG subclass (IgG1 and IgG2a) antibodies in sera from the vaccinated animals after 1:100 dilution were determined by ELISA. The mean OD405 values of pooled sera from each group (5 mice per group) were presented. The background OD405 of normal mouse sera was
    Figure Legend Snippet: Vaccination with scFv N418 -HER2 induced HER2-specific cellular and antibody immune response. BALB/c mice were vaccinated with HER2, scFv N418 -neu or scFv N418 -HER2. Control animals received pcDNA3.1. The draining lymph nodes and spleens were harvested from the vaccinated animals after two vaccinations. A. CD4 + T cells isolated from the draining lymph were cultured in the presence of 10 µg/ml recombinant HER2 or TRP2 protein for 4 d with the addition of [ 3 H] thymidine in the last 16 h. T-cell proliferation was determined by [ 3 H] thymidine incorporation (left panel). Right panel, the supernatant recovered from the assay in left was tested for cytokine production by ELISA. B. Splenocytes isolated from the vaccinated animals were stimulated for 6 h with H-2K d -restricted HER2 63-71 peptide TYLPTNASL before flow cytometric analysis with anti-CD8 and anti-IFN-γ antibodies. Left, increase in CD8 + IFN-γ + cells upon peptide stimulation of splenocytes. Representative results from one animal of scFv N418 -HER2 group upon restimulation in the presence of HER2 or TRP2 peptide. Right, absolute numbers of CD8 + IFN-γ + splenocytes (mean values from five mice per group). C. Splenocytes were cocultured with D2F2/E2 cells for 5 d. The resultant splenocytes (E) were cocultured for 4 h with the 51 Cr-labeled target cells (T). Percentages of target cells killing by the splenocytes from the vaccinated mice are shown. Data represent the means of triplicate cultures and are representative of two independent experiments. D. HER2-specific total IgG and IgG subclass (IgG1 and IgG2a) antibodies in sera from the vaccinated animals after 1:100 dilution were determined by ELISA. The mean OD405 values of pooled sera from each group (5 mice per group) were presented. The background OD405 of normal mouse sera was

    Techniques Used: Mouse Assay, Isolation, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Labeling

    Vaccination with scFv N418 -HER2 protects mice from challenge with HER2-expressing tumor cells and induces memory immune responses. A. Animals (10 mice per group) were vaccinated with HER2, neu, scFv N418 -HER2 or scFv N418 -neu on days -21 and -7. Control animals received pcDNA3.1. On day 0, mice were inoculated s.c. with D2F2/E2 tumor cells. Tumor developments were monitored, and animal survival was calculated. Left panel, kinetics of tumor growth; Right panel, survival curve. B. The vaccinated animals were challenged with parental HER2-negative D2F2 cells on day 0, Left panel, kinetics of tumor growth; Right panel, survival curve. C. The long-term surviving mice from scFv N418 -HER2 group were rechallenged with D2F2/E2, D2F2, or syngeneic unrelated 4T1 tumor cells 3 months after initial tumor challenge. D. Naive mice injected s.c. with D2F2/E2, D2F2, or 4T1 tumor cells served as a control. Tumor growth was followed by caliper measurements, and results are represented as the mean tumor volume (mm 3 ). The data were representative of two experiments with comparable results. Bars, SE. *P
    Figure Legend Snippet: Vaccination with scFv N418 -HER2 protects mice from challenge with HER2-expressing tumor cells and induces memory immune responses. A. Animals (10 mice per group) were vaccinated with HER2, neu, scFv N418 -HER2 or scFv N418 -neu on days -21 and -7. Control animals received pcDNA3.1. On day 0, mice were inoculated s.c. with D2F2/E2 tumor cells. Tumor developments were monitored, and animal survival was calculated. Left panel, kinetics of tumor growth; Right panel, survival curve. B. The vaccinated animals were challenged with parental HER2-negative D2F2 cells on day 0, Left panel, kinetics of tumor growth; Right panel, survival curve. C. The long-term surviving mice from scFv N418 -HER2 group were rechallenged with D2F2/E2, D2F2, or syngeneic unrelated 4T1 tumor cells 3 months after initial tumor challenge. D. Naive mice injected s.c. with D2F2/E2, D2F2, or 4T1 tumor cells served as a control. Tumor growth was followed by caliper measurements, and results are represented as the mean tumor volume (mm 3 ). The data were representative of two experiments with comparable results. Bars, SE. *P

    Techniques Used: Mouse Assay, Expressing, Injection

    Protective effects of scFv N418 -neu in transgenic BALB-neuT mice. A. Female BALB-neuT mice (10 mice per group) were vaccinated with neu, HER2, scFv N418 -HER2 or scFv N418 -neu in left hind limb on days -21 and -7. Control animals received pcDNA3.1. On day 0, mice were inoculated s.c. with neu-expressing TUBO cells in opposite flank. Left panel, kinetics of tumor growth; Right panel, survival curve. *P
    Figure Legend Snippet: Protective effects of scFv N418 -neu in transgenic BALB-neuT mice. A. Female BALB-neuT mice (10 mice per group) were vaccinated with neu, HER2, scFv N418 -HER2 or scFv N418 -neu in left hind limb on days -21 and -7. Control animals received pcDNA3.1. On day 0, mice were inoculated s.c. with neu-expressing TUBO cells in opposite flank. Left panel, kinetics of tumor growth; Right panel, survival curve. *P

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing

    21) Product Images from "Establishment of stable Huh-7 cell lines expressing various hepatitis C virus genotype 3a protein: an in-vitro testing system for novel anti-HCV drugs"

    Article Title: Establishment of stable Huh-7 cell lines expressing various hepatitis C virus genotype 3a protein: an in-vitro testing system for novel anti-HCV drugs

    Journal: Genetic Vaccines and Therapy

    doi: 10.1186/1479-0556-9-12

    a) Showing the amplified genes of Core (573), Envelope 1(576), Non-structural 2 (NS2, 642 bp) and Non Structural 4a (NS4A, 168 bp), b) lanes 2-5 (left to right) showing the complete amplified region of 783 bp of Non Structural 4b gene . From the HCV positive serum with 3a genotype, RNA was extracted and individual gene was reverse transcribed using M-MLV. HCV reference sequence of NZL1 # D17763 was used for primer designing on Primer 3 software, restriction sites and kozak sequences were added after restriction analysis on web cutter and neb-cutter primers sequences. Each entire gene was amplified individually. Amplified genes with restriction sites were then cloned in mammalian expression vector PcDNA3.1+ .
    Figure Legend Snippet: a) Showing the amplified genes of Core (573), Envelope 1(576), Non-structural 2 (NS2, 642 bp) and Non Structural 4a (NS4A, 168 bp), b) lanes 2-5 (left to right) showing the complete amplified region of 783 bp of Non Structural 4b gene . From the HCV positive serum with 3a genotype, RNA was extracted and individual gene was reverse transcribed using M-MLV. HCV reference sequence of NZL1 # D17763 was used for primer designing on Primer 3 software, restriction sites and kozak sequences were added after restriction analysis on web cutter and neb-cutter primers sequences. Each entire gene was amplified individually. Amplified genes with restriction sites were then cloned in mammalian expression vector PcDNA3.1+ .

    Techniques Used: Amplification, Sequencing, Software, Clone Assay, Expressing, Plasmid Preparation

    22) Product Images from "Thioredoxin Confers Intrinsic Resistance to Cytostatic Drugs in Human Glioma Cells"

    Article Title: Thioredoxin Confers Intrinsic Resistance to Cytostatic Drugs in Human Glioma Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19102874

    Overexpression of the tumour suppressor TXNIP inhibits Trx expression and sensitizes GBM cell lines to cisplatin. ( a ) Representative Western blots of U87 cells showing overexpression of TXNIP and downregulation of Trx-1 protein expression after transient transfection with pcDNA3.1-TXNIP or empty vector (pcDNA3.1) 48 h post-transfection. β-actin Western blot was performed to control for loading; ( b – d ) MTT assays with cisplatin concentration-response curves showing sensitization of U87 ( b ), U251 ( c ), and U373 ( d ) cells after transient transfection with empty vector (pcDNA3.1) or pcDNA3.1-TXNIP. Cells were incubated with cisplatin for 72 h at 24 h post-transfection; ( e ) Respective cisplatin IC 50 values derived from curves presented in b – d ( n = 4, * p
    Figure Legend Snippet: Overexpression of the tumour suppressor TXNIP inhibits Trx expression and sensitizes GBM cell lines to cisplatin. ( a ) Representative Western blots of U87 cells showing overexpression of TXNIP and downregulation of Trx-1 protein expression after transient transfection with pcDNA3.1-TXNIP or empty vector (pcDNA3.1) 48 h post-transfection. β-actin Western blot was performed to control for loading; ( b – d ) MTT assays with cisplatin concentration-response curves showing sensitization of U87 ( b ), U251 ( c ), and U373 ( d ) cells after transient transfection with empty vector (pcDNA3.1) or pcDNA3.1-TXNIP. Cells were incubated with cisplatin for 72 h at 24 h post-transfection; ( e ) Respective cisplatin IC 50 values derived from curves presented in b – d ( n = 4, * p

    Techniques Used: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, MTT Assay, Concentration Assay, Incubation, Derivative Assay

    23) Product Images from "Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-?B and proinflammatory gene program activation in intestinal epithelial cells"

    Article Title: Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-?B and proinflammatory gene program activation in intestinal epithelial cells

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-4-33

    TLR5 inhibits flagellin-mediated NF-κB reporter gene activity. HT29 cells were transfected in triplicate in 6-well dishes using the indicated DN-TLR mammalian expression vectors or antisense TLR5 (AS TLR5) (2 μg/well), 2× NF-κB Luc reporter gene (100 ng/well), pRL-TK Renilla luciferase for normalization (50 ng/well) adjusted to 4 μg total DNA/well with empty vector pCDNA3.1 DNA. A, Fold-induction of 2× NF-κB Luc reporter gene in non-stimulated cells (light shading) and in TNFα (10 ng/ml) treated cells (dark shading). Lysates were prepared 12 h after stimulation. Results of a representative experiment are shown. B, HT29 cells transfected as in A were treated with flagellin (1 μg/ml) and cell lysates were prepared and analyzed as in Fig. 8A. Results of a representative experiment are shown.
    Figure Legend Snippet: TLR5 inhibits flagellin-mediated NF-κB reporter gene activity. HT29 cells were transfected in triplicate in 6-well dishes using the indicated DN-TLR mammalian expression vectors or antisense TLR5 (AS TLR5) (2 μg/well), 2× NF-κB Luc reporter gene (100 ng/well), pRL-TK Renilla luciferase for normalization (50 ng/well) adjusted to 4 μg total DNA/well with empty vector pCDNA3.1 DNA. A, Fold-induction of 2× NF-κB Luc reporter gene in non-stimulated cells (light shading) and in TNFα (10 ng/ml) treated cells (dark shading). Lysates were prepared 12 h after stimulation. Results of a representative experiment are shown. B, HT29 cells transfected as in A were treated with flagellin (1 μg/ml) and cell lysates were prepared and analyzed as in Fig. 8A. Results of a representative experiment are shown.

    Techniques Used: Activity Assay, Transfection, Expressing, Luciferase, Plasmid Preparation

    24) Product Images from "Long non-coding RNA HOTAIR, a c-Myc activated driver of malignancy, negatively regulates miRNA-130a in gallbladder cancer"

    Article Title: Long non-coding RNA HOTAIR, a c-Myc activated driver of malignancy, negatively regulates miRNA-130a in gallbladder cancer

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-13-156

    The expression changes of c-Myc or HOTAIR after transfection of c-Myc siRNA or pcDNA3.1-c-Myc in GBC-SD cells. The relative mRNA expression levels were evaluated with real-time qPCR. c-Myc protein levels were determined using Western blot assay. Each experiment was performed in triplicate. (A) pcDNA3.1-c-Myc markedly upregulated the expression of c-Myc at mRNA levels. (B) Representative images of western blot results indicated pcDNA3.1-c-Myc significantly upregulated the expression of c-Myc at protein levels. (C) pcDNA3.1-c-Myc significantly upregulated the expression of HOTAIR at mRNA levels. (D) c-Myc siRNA significantly downregulated the expression of c-Myc at mRNA levels. (E) ) Representative images of western blot results indicated c-Myc siRNA significantly downregulated the expression of c-Myc at protein level. (F) c-Myc siRNA significantly down-regulated the expression of HOTAIR at mRNA levels. Error bars represent S.E.M., n = 3. * p
    Figure Legend Snippet: The expression changes of c-Myc or HOTAIR after transfection of c-Myc siRNA or pcDNA3.1-c-Myc in GBC-SD cells. The relative mRNA expression levels were evaluated with real-time qPCR. c-Myc protein levels were determined using Western blot assay. Each experiment was performed in triplicate. (A) pcDNA3.1-c-Myc markedly upregulated the expression of c-Myc at mRNA levels. (B) Representative images of western blot results indicated pcDNA3.1-c-Myc significantly upregulated the expression of c-Myc at protein levels. (C) pcDNA3.1-c-Myc significantly upregulated the expression of HOTAIR at mRNA levels. (D) c-Myc siRNA significantly downregulated the expression of c-Myc at mRNA levels. (E) ) Representative images of western blot results indicated c-Myc siRNA significantly downregulated the expression of c-Myc at protein level. (F) c-Myc siRNA significantly down-regulated the expression of HOTAIR at mRNA levels. Error bars represent S.E.M., n = 3. * p

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

    c-Myc regulates HOTAIR promoter activity, depending on E-box element. (A) Dual luciferase assay on GBC-SD cells cotransfected with firefly luciferase constructs containing the HOTAIR promoter and pcDNA3.1 or pcDNA3.1-c-Myc. (B) Dual luciferase assay on GBC-SD cells cotransfected with firefly luciferase constructs containing the HOTAIR promoter and c-Myc siRNA or the control siRNA. (C) schematic of the HOTAIR-promoter-luciferase construct is depicted with locations of the E-box element and sequences of point mutation. (D, E) Dual luciferase assay on GBC-SD cells cotransfected with firefly luciferase constructs (mutant at E-box element) and pcDNA3.1-c-Myc (D) or c-Myc siRNA (E) . All of the transfection was performed in triplicates. The values are presented as the mean ± S.E.M. of the ratio of firefly luciferase activity to renilla luciferase activity and are representative of at least three independent experiments. Data are shown as the mean ± S.E.M, based on at least three independent experiments. * p
    Figure Legend Snippet: c-Myc regulates HOTAIR promoter activity, depending on E-box element. (A) Dual luciferase assay on GBC-SD cells cotransfected with firefly luciferase constructs containing the HOTAIR promoter and pcDNA3.1 or pcDNA3.1-c-Myc. (B) Dual luciferase assay on GBC-SD cells cotransfected with firefly luciferase constructs containing the HOTAIR promoter and c-Myc siRNA or the control siRNA. (C) schematic of the HOTAIR-promoter-luciferase construct is depicted with locations of the E-box element and sequences of point mutation. (D, E) Dual luciferase assay on GBC-SD cells cotransfected with firefly luciferase constructs (mutant at E-box element) and pcDNA3.1-c-Myc (D) or c-Myc siRNA (E) . All of the transfection was performed in triplicates. The values are presented as the mean ± S.E.M. of the ratio of firefly luciferase activity to renilla luciferase activity and are representative of at least three independent experiments. Data are shown as the mean ± S.E.M, based on at least three independent experiments. * p

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

    Identification of miR-130a as a target of HOTAIR. (A) pcDNA3.1-HOTAIR upregulated the HOTAIR mRNA level. (B, C) Downregulation of miR-130a by ectopic expression of HOTAIR detected by RT-PCR (B) and northern blot (C) . GBC-SD cells were transfected with vector control or HOTAIR or mutant HOTAIR, and total RNA was isolated 48 h after transfection. (D) The mutant HOTAIR at putative binding site. Error bars represent S.E.M., n = 3. * p
    Figure Legend Snippet: Identification of miR-130a as a target of HOTAIR. (A) pcDNA3.1-HOTAIR upregulated the HOTAIR mRNA level. (B, C) Downregulation of miR-130a by ectopic expression of HOTAIR detected by RT-PCR (B) and northern blot (C) . GBC-SD cells were transfected with vector control or HOTAIR or mutant HOTAIR, and total RNA was isolated 48 h after transfection. (D) The mutant HOTAIR at putative binding site. Error bars represent S.E.M., n = 3. * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Transfection, Plasmid Preparation, Mutagenesis, Isolation, Binding Assay

    25) Product Images from "The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation *"

    Article Title: The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.495754

    Constitutive internalization of SERT is dynamin-dependent. CAD cells were co-transfected with TacSERT and wild type dynamin I, dominant negative (K44A) dynamin I, or the empty vector pcDNA3.1. A , intracellular accumulation of TacSERT measured in the ELISA-based
    Figure Legend Snippet: Constitutive internalization of SERT is dynamin-dependent. CAD cells were co-transfected with TacSERT and wild type dynamin I, dominant negative (K44A) dynamin I, or the empty vector pcDNA3.1. A , intracellular accumulation of TacSERT measured in the ELISA-based

    Techniques Used: Transfection, Dominant Negative Mutation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    HA-SERT is internalized via a dynamin I-dependent pathway and co-localizes with Rab4 and Rab7. A , CAD cells co-transfected with HA-SERT and wild type dynamin I, dominant negative (K44A) dynamin I, or the empty vector pcDNA3.1. B , quantifications of co-localizations
    Figure Legend Snippet: HA-SERT is internalized via a dynamin I-dependent pathway and co-localizes with Rab4 and Rab7. A , CAD cells co-transfected with HA-SERT and wild type dynamin I, dominant negative (K44A) dynamin I, or the empty vector pcDNA3.1. B , quantifications of co-localizations

    Techniques Used: Transfection, Dominant Negative Mutation, Plasmid Preparation

    26) Product Images from "Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency"

    Article Title: Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddv074

    Long-chain FAO and CI defects are restored in ACAD9 -deficient cells by transfection of an ACAD9 expression vector. ( A ) Re-expression of ACAD9 in ACAD9 −/− HEK293 clone A cells transfected with empty pcDNA3.1 vector (Vect) or pcDNA-ACAD9 (ACAD9). The ACAD9 antigen was visualized with green fluorescently tagged antibodies, and mitochondrial cytochrome c oxidase (COX) was visualized with red fluorescently tagged antibodies. The merged image (arrow) shows co-localization of ACAD9 and COX in mitochondria as yellow. Scale bar, 10.75 μm. ( B ) Etomoxir-sensitive palmitate oxidation rates were determined in pcDNA3.1 (Vect) and pcDNA-ACAD9 (ACAD9) transfected clone A. Values are presented as average ± SD ( n = 4); * P
    Figure Legend Snippet: Long-chain FAO and CI defects are restored in ACAD9 -deficient cells by transfection of an ACAD9 expression vector. ( A ) Re-expression of ACAD9 in ACAD9 −/− HEK293 clone A cells transfected with empty pcDNA3.1 vector (Vect) or pcDNA-ACAD9 (ACAD9). The ACAD9 antigen was visualized with green fluorescently tagged antibodies, and mitochondrial cytochrome c oxidase (COX) was visualized with red fluorescently tagged antibodies. The merged image (arrow) shows co-localization of ACAD9 and COX in mitochondria as yellow. Scale bar, 10.75 μm. ( B ) Etomoxir-sensitive palmitate oxidation rates were determined in pcDNA3.1 (Vect) and pcDNA-ACAD9 (ACAD9) transfected clone A. Values are presented as average ± SD ( n = 4); * P

    Techniques Used: Transfection, Expressing, Plasmid Preparation

    27) Product Images from "Restriction of HIV-1 Replication in Monocytes Is Abolished by Vpx of SIVsmmPBj"

    Article Title: Restriction of HIV-1 Replication in Monocytes Is Abolished by Vpx of SIVsmmPBj

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007098

    Schematic representation of expression constructs and packaging of Vpr/Vpx fusion proteins into vector particles was detected by Western-blotting. (A) The viral proteins HIV-1 Vpr, SIVsmmPBj1.9 Vpx and related fusion proteins encoded by the expression vector pcDNA3.1. For generation of the fusion protein, the full length Vpx was ligated to HIV-1 Vpr. HA, hemagglutinin tag. (B) HIV-1- or SIVsmmPBj- derived transfer vectors coding for EGFP. In the PBj4xko-EGFP vector, coding regions of all four accessory genes are knocked-out by insertion of stop-codons marked by an asterisk. (C) PBj- and HIV-1-derived packaging constructs coding for Gag-Pol and the regulatory proteins Tat and Rev. These constructs were also used for production of virus-like particles. For pseudotyping of vectors or virus-like particles, the vesicular stomatitis virus G protein (VSV-G, not shown) was used. (D) Western blots of supernatants or lysates of 293T packaging cells cotransfected with the respective vector constructs required for generation of VSV-G-pseudotyped lentiviral vectors (HIV-1 LV or PBj4xko LV), and one of the expression constructs encoding Vpr, Vpx or the Vpr/Vpx fusion protein as indicated on the top. As controls, supernatants and lysates of cells transfected only with one of the Vpr, Vpx or Vpr/Vpx constructs were analyzed. Cell supernatants were purified by filtering and sucrose cushion centrifugation. For labelling, antibodies directed against HA, Vpx, or the envelope protein VSV-G were used. Ψ, packaging signal; LTR, long terminal repeat; SD, splice donor; cPPT, central polypurine tract; RRE, rev responsive element; SA, splice acceptor; CMV, cytomegalovirus promoter; BGHpA, bovine growth hormone polyadenylation signal.
    Figure Legend Snippet: Schematic representation of expression constructs and packaging of Vpr/Vpx fusion proteins into vector particles was detected by Western-blotting. (A) The viral proteins HIV-1 Vpr, SIVsmmPBj1.9 Vpx and related fusion proteins encoded by the expression vector pcDNA3.1. For generation of the fusion protein, the full length Vpx was ligated to HIV-1 Vpr. HA, hemagglutinin tag. (B) HIV-1- or SIVsmmPBj- derived transfer vectors coding for EGFP. In the PBj4xko-EGFP vector, coding regions of all four accessory genes are knocked-out by insertion of stop-codons marked by an asterisk. (C) PBj- and HIV-1-derived packaging constructs coding for Gag-Pol and the regulatory proteins Tat and Rev. These constructs were also used for production of virus-like particles. For pseudotyping of vectors or virus-like particles, the vesicular stomatitis virus G protein (VSV-G, not shown) was used. (D) Western blots of supernatants or lysates of 293T packaging cells cotransfected with the respective vector constructs required for generation of VSV-G-pseudotyped lentiviral vectors (HIV-1 LV or PBj4xko LV), and one of the expression constructs encoding Vpr, Vpx or the Vpr/Vpx fusion protein as indicated on the top. As controls, supernatants and lysates of cells transfected only with one of the Vpr, Vpx or Vpr/Vpx constructs were analyzed. Cell supernatants were purified by filtering and sucrose cushion centrifugation. For labelling, antibodies directed against HA, Vpx, or the envelope protein VSV-G were used. Ψ, packaging signal; LTR, long terminal repeat; SD, splice donor; cPPT, central polypurine tract; RRE, rev responsive element; SA, splice acceptor; CMV, cytomegalovirus promoter; BGHpA, bovine growth hormone polyadenylation signal.

    Techniques Used: Expressing, Construct, Plasmid Preparation, Western Blot, Derivative Assay, Transfection, Purification, Centrifugation

    28) Product Images from "Comparative analysis of the immunogenicity of SARS-CoV nucleocapsid DNA vaccine administrated with different routes in mouse model"

    Article Title: Comparative analysis of the immunogenicity of SARS-CoV nucleocapsid DNA vaccine administrated with different routes in mouse model

    Journal: Vaccine

    doi: 10.1016/j.vaccine.2009.01.021

    N-specific lymphocyte proliferation assay. Pooled splenocytes were obtained from mice (5 mice per group) on day 10 after the final immunization. Mice immunized with the DNA vaccine intramuscularly (i.m.), by electroporation (i.e.), orally using live-attenuated Salmonella typhimurium (oral). Splenocytes were stimulated in vitro with N protein (test groups), Con A (positive controls), and BSA (irrelevant antigen controls). Splenocytes from the control groups (CS022, pcDNA3.1, or PBS) were stimulated with N protein, and served as negative controls and sham controls. The stimulation index (SI) was calculated using the following formula: SI = (mean OD of Con A- or antigen-stimulated proliferation)/(mean OD of non-stimulated proliferation). Each bar represents the mean SI ± S.D. of 5 mice.
    Figure Legend Snippet: N-specific lymphocyte proliferation assay. Pooled splenocytes were obtained from mice (5 mice per group) on day 10 after the final immunization. Mice immunized with the DNA vaccine intramuscularly (i.m.), by electroporation (i.e.), orally using live-attenuated Salmonella typhimurium (oral). Splenocytes were stimulated in vitro with N protein (test groups), Con A (positive controls), and BSA (irrelevant antigen controls). Splenocytes from the control groups (CS022, pcDNA3.1, or PBS) were stimulated with N protein, and served as negative controls and sham controls. The stimulation index (SI) was calculated using the following formula: SI = (mean OD of Con A- or antigen-stimulated proliferation)/(mean OD of non-stimulated proliferation). Each bar represents the mean SI ± S.D. of 5 mice.

    Techniques Used: Lymphocyte Proliferation Assay, Mouse Assay, Electroporation, In Vitro

    Analysis the recombinant SARS-CoV N protein expression in vitro by Western blot. The expression of SARS-CoV N protein was determined in 293 cells transfected with pcDNA-N, or pcDNA3.1(+) by Western blot analysis. Rat anti-N-specific antibody was used at a 1:100 dilution for the detection of N protein expression. Lane 1: lysate of 293T cells transfected with pcDNA-N; Lane 2: lysate of 293T cells transfected with pcDNA3.1(+); Lane 3: Protein molecular weight marker (Gibco).
    Figure Legend Snippet: Analysis the recombinant SARS-CoV N protein expression in vitro by Western blot. The expression of SARS-CoV N protein was determined in 293 cells transfected with pcDNA-N, or pcDNA3.1(+) by Western blot analysis. Rat anti-N-specific antibody was used at a 1:100 dilution for the detection of N protein expression. Lane 1: lysate of 293T cells transfected with pcDNA-N; Lane 2: lysate of 293T cells transfected with pcDNA3.1(+); Lane 3: Protein molecular weight marker (Gibco).

    Techniques Used: Recombinant, Expressing, In Vitro, Western Blot, Transfection, Molecular Weight, Marker

    29) Product Images from "FoxQ1 Promotes Glioma Cells Proliferation and Migration by Regulating NRXN3 Expression"

    Article Title: FoxQ1 Promotes Glioma Cells Proliferation and Migration by Regulating NRXN3 Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0055693

    The NRXN3 as a transcriptional target of FoxQ1. A , Sequence and position of putative FoxQ1 binding sites on the NRXN3 promoter. B , ChIP assays were done with U-87MG cells. Chromatin fragments of the cells were immunoprecipitated with anti-FoxQ1 antibody or negative control IgG (middle) and subjected to PCR. We subjected 1% of the total cell lysates to PCR before immunoprecipitation as inputs. C , schematic structure of the NRXN3 promoter. The sequence of the FoxQ1 binding sites are shown in both wild-type (WT) and mutant (Mut) forms. D+E , Luciferase activity with or without mutations in NRXN3 promoter. U-87MG cells were transfected with the wild-type NRXN3 promoter or its mutants (D). SW1088 cells were co-transfected with the wild-type NRXN3 promoter or its mutants and pcDNA3.1-FoxQ1 (E). Luciferase activities were then determined. Three independent experiments were conducted. * P
    Figure Legend Snippet: The NRXN3 as a transcriptional target of FoxQ1. A , Sequence and position of putative FoxQ1 binding sites on the NRXN3 promoter. B , ChIP assays were done with U-87MG cells. Chromatin fragments of the cells were immunoprecipitated with anti-FoxQ1 antibody or negative control IgG (middle) and subjected to PCR. We subjected 1% of the total cell lysates to PCR before immunoprecipitation as inputs. C , schematic structure of the NRXN3 promoter. The sequence of the FoxQ1 binding sites are shown in both wild-type (WT) and mutant (Mut) forms. D+E , Luciferase activity with or without mutations in NRXN3 promoter. U-87MG cells were transfected with the wild-type NRXN3 promoter or its mutants (D). SW1088 cells were co-transfected with the wild-type NRXN3 promoter or its mutants and pcDNA3.1-FoxQ1 (E). Luciferase activities were then determined. Three independent experiments were conducted. * P

    Techniques Used: Sequencing, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Mutagenesis, Luciferase, Activity Assay, Transfection

    Effect of FoxQ1/NRXN3 expression on proliferation and migration of glioma cells in vitro . A+D , Western blot (upper) and RT-qPCR (lower) analyses of FoxQ1 and NRXN3 expression in stable FoxQ1shRNA-transfected U-87MG cells (A) and pcDNA3.1-FoxQ1-transfected SW1088 cells (D). B+E , Cells as in (A) or (B) were cultured in 96-well plates and analyzed by MTT assay. Cell proliferation curves were shown in 9 days. Three independent experiments were conducted. C+F , Cells as in (A) or (B) were examined for cell migration motility in 24-well plates with transwell chambers. Migrated cells were stained with crystal violet and counted under a light microscope. Three independent experiments were conducted. * P
    Figure Legend Snippet: Effect of FoxQ1/NRXN3 expression on proliferation and migration of glioma cells in vitro . A+D , Western blot (upper) and RT-qPCR (lower) analyses of FoxQ1 and NRXN3 expression in stable FoxQ1shRNA-transfected U-87MG cells (A) and pcDNA3.1-FoxQ1-transfected SW1088 cells (D). B+E , Cells as in (A) or (B) were cultured in 96-well plates and analyzed by MTT assay. Cell proliferation curves were shown in 9 days. Three independent experiments were conducted. C+F , Cells as in (A) or (B) were examined for cell migration motility in 24-well plates with transwell chambers. Migrated cells were stained with crystal violet and counted under a light microscope. Three independent experiments were conducted. * P

    Techniques Used: Expressing, Migration, In Vitro, Western Blot, Quantitative RT-PCR, Transfection, Cell Culture, MTT Assay, Staining, Light Microscopy

    30) Product Images from "Increased Furin Activity Enhances the Malignant Phenotype of Human Head and Neck Cancer Cells"

    Article Title: Increased Furin Activity Enhances the Malignant Phenotype of Human Head and Neck Cancer Cells

    Journal: The American Journal of Pathology

    doi:

    Western blot analysis of furin expression in cells transfected with vector-alone (pcDNA3.1) or full-length furin cDNA (pcDNA3.1 fur). Vertical arrows indicate clones selected for further studies. A: SCC12. B: SCC15. C: Western analysis of MT1-MMP showing the processed form (60 kd, full arrow ) and the pro-form (63 kd, arrowhead ). Cell lysates from SCC12 cells (100 μg) and SCC15 (30 μg) were fractionated in 10% SDS-PAGE. Note the higher proportion of processed form in the furin-transfected cells.
    Figure Legend Snippet: Western blot analysis of furin expression in cells transfected with vector-alone (pcDNA3.1) or full-length furin cDNA (pcDNA3.1 fur). Vertical arrows indicate clones selected for further studies. A: SCC12. B: SCC15. C: Western analysis of MT1-MMP showing the processed form (60 kd, full arrow ) and the pro-form (63 kd, arrowhead ). Cell lysates from SCC12 cells (100 μg) and SCC15 (30 μg) were fractionated in 10% SDS-PAGE. Note the higher proportion of processed form in the furin-transfected cells.

    Techniques Used: Western Blot, Expressing, Transfection, Plasmid Preparation, Clone Assay, SDS Page

    A: Gelatinase zymogram of SCC12 and SCC15 vector-alone (pcDNA3.1) and furin (pcDNA3.1 fur)-transfected cells. B: Histogram of the in vitro invasion assays performed with furin transfectants from two HNSCC lines and their respective vector-alone-transfected cells. Results are expressed as number of cells per field. Each bar represents a mean of three different experiments. An average of 4000 cells was counted per transfected clone. White bars: vector-alone-transfected cells; black bars: furin transfectants. One-sided t -test indicated statistically significant differences ( P
    Figure Legend Snippet: A: Gelatinase zymogram of SCC12 and SCC15 vector-alone (pcDNA3.1) and furin (pcDNA3.1 fur)-transfected cells. B: Histogram of the in vitro invasion assays performed with furin transfectants from two HNSCC lines and their respective vector-alone-transfected cells. Results are expressed as number of cells per field. Each bar represents a mean of three different experiments. An average of 4000 cells was counted per transfected clone. White bars: vector-alone-transfected cells; black bars: furin transfectants. One-sided t -test indicated statistically significant differences ( P

    Techniques Used: Plasmid Preparation, Transfection, In Vitro

    A: Subcutaneous tumorigenicity of SCC15 transfectant cells after inoculation in the dorsal s.c. tissue of Scid mice. Squares , vector-alone (pcDNA3.1)-transfected cells; rhombus , furin-transfected cells. One-sided t -test indicated statistically significant differences ( P
    Figure Legend Snippet: A: Subcutaneous tumorigenicity of SCC15 transfectant cells after inoculation in the dorsal s.c. tissue of Scid mice. Squares , vector-alone (pcDNA3.1)-transfected cells; rhombus , furin-transfected cells. One-sided t -test indicated statistically significant differences ( P

    Techniques Used: Transfection, Mouse Assay, Plasmid Preparation

    31) Product Images from "DNA vaccines targeting the encoded antigens to dendritic cells induce potent antitumor immunity in mice"

    Article Title: DNA vaccines targeting the encoded antigens to dendritic cells induce potent antitumor immunity in mice

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-14-39

    Vaccination with scFv NLDC-145 -HER2 protects mice from challenge with HER2-expressing tumor cells and induces memory immune responses. A animals (10 mice per group) were vaccinated with HER2, neu, scFv NLDC-145 -HER2 or scFv NLDC-145 -neu on days -21 and -7. Control animals received pcDNA3.1. On day 0, mice were inoculated s.c. with D2F2/E2 tumor cells. Tumor developments were monitored, and animal survival was calculated. Left panel , kinetics of tumor growth; Right panel , survival curve. B the vaccinated animals were challenged with parental HER2-negative D2F2 cells on day 0. C the long-term surviving mice from scFv NLDC-145 -HER2 group were rechallenged with D2F2/E2, D2F2, or syngeneic unrelated 4 T1 tumor cells 3 months after initial tumor challenge. Tumor growth was followed. Bars, SE. *, P
    Figure Legend Snippet: Vaccination with scFv NLDC-145 -HER2 protects mice from challenge with HER2-expressing tumor cells and induces memory immune responses. A animals (10 mice per group) were vaccinated with HER2, neu, scFv NLDC-145 -HER2 or scFv NLDC-145 -neu on days -21 and -7. Control animals received pcDNA3.1. On day 0, mice were inoculated s.c. with D2F2/E2 tumor cells. Tumor developments were monitored, and animal survival was calculated. Left panel , kinetics of tumor growth; Right panel , survival curve. B the vaccinated animals were challenged with parental HER2-negative D2F2 cells on day 0. C the long-term surviving mice from scFv NLDC-145 -HER2 group were rechallenged with D2F2/E2, D2F2, or syngeneic unrelated 4 T1 tumor cells 3 months after initial tumor challenge. Tumor growth was followed. Bars, SE. *, P

    Techniques Used: Mouse Assay, Expressing

    Vaccination with scFv NLDC-145 -HER2 induced HER2-specific cellular immune response. BALB/c mice were vaccinated with HER2, scFv NLDC-145 -neu or scFv NLDC-145 -HER2. Control animals received PBS or pcDNA3.1. Spleens and peripheral blood were harvested from the vaccinated animals after two vaccinations. A splenocytes isolated from the vaccinated animals were cultured in the presence of 10 μg/mL recombinant HER2 or TRP2 protein for 4 d with the addition of [ 3 H] thymidine in the last 16 h. T-cell proliferation was determined by [ 3 H] thymidine incorporation ( left panel ). Right panel , the supernatant recovered from the assay in left was tested for cytokine production by ELISA. B intracellular staining for IFN-γ and TNF-α in splenocytes from the vaccinated animals stimulated with 10 μg/mL recombinant HER2 or TRP2 protein for 24 h and brefeldin A added during the final 4 h of incubation. One representative dot plots from scFv NLDC-145 -HER2-vaccinated animal. C percentage of CD4 + TNF-α + , CD4 + IFN-γ + , CD8 + TNF-α + and CD8 + IFN-γ + cells in total CD4 + and CD 8 + T cells from each group are shown. The data are mean percentages ± SE. D splenocytes were cocultured with D2F2/E2 cells for 5 d. The resultant splenocytes (E) were cocultured for 4 h with the 51 Cr-labeled target cells (T) ( left panel ). Right panel , the restimulated splenocytes from scFv NLDC-145 -HER2 vaccinated mice were also cocultured for 4 h with the 51 Cr-labeled D2F2 or EL4/E2 (different gene background control) target cells. Percentages of target cells killing by the splenocytes from the vaccinated mice are shown. Data represent the means of triplicate cultures and are representative of two independent experiments. Bars, SE. *, P
    Figure Legend Snippet: Vaccination with scFv NLDC-145 -HER2 induced HER2-specific cellular immune response. BALB/c mice were vaccinated with HER2, scFv NLDC-145 -neu or scFv NLDC-145 -HER2. Control animals received PBS or pcDNA3.1. Spleens and peripheral blood were harvested from the vaccinated animals after two vaccinations. A splenocytes isolated from the vaccinated animals were cultured in the presence of 10 μg/mL recombinant HER2 or TRP2 protein for 4 d with the addition of [ 3 H] thymidine in the last 16 h. T-cell proliferation was determined by [ 3 H] thymidine incorporation ( left panel ). Right panel , the supernatant recovered from the assay in left was tested for cytokine production by ELISA. B intracellular staining for IFN-γ and TNF-α in splenocytes from the vaccinated animals stimulated with 10 μg/mL recombinant HER2 or TRP2 protein for 24 h and brefeldin A added during the final 4 h of incubation. One representative dot plots from scFv NLDC-145 -HER2-vaccinated animal. C percentage of CD4 + TNF-α + , CD4 + IFN-γ + , CD8 + TNF-α + and CD8 + IFN-γ + cells in total CD4 + and CD 8 + T cells from each group are shown. The data are mean percentages ± SE. D splenocytes were cocultured with D2F2/E2 cells for 5 d. The resultant splenocytes (E) were cocultured for 4 h with the 51 Cr-labeled target cells (T) ( left panel ). Right panel , the restimulated splenocytes from scFv NLDC-145 -HER2 vaccinated mice were also cocultured for 4 h with the 51 Cr-labeled D2F2 or EL4/E2 (different gene background control) target cells. Percentages of target cells killing by the splenocytes from the vaccinated mice are shown. Data represent the means of triplicate cultures and are representative of two independent experiments. Bars, SE. *, P

    Techniques Used: Mouse Assay, Isolation, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Incubation, Labeling

    Vaccination with scFv NLDC-145 -HER2 induced HER2-specific antibodies. BALB/c mice were vaccinated with HER2, scFv NLDC-145 -neu or scFv NLDC-145 -HER2. Control animals received PBS or pcDNA3.1. Sera were collected from the vaccinated animals after two vaccinations. A HER2-specific total IgG and IgG subclass (IgG1and IgG2a) antibodies in sera from the vaccinated animals after 1:100 dilution were determined by ELISA. The mean OD405 values of pooled sera from each group (5 mice per group) were presented. Bars, SE. The background OD405 of normal mouse sera was
    Figure Legend Snippet: Vaccination with scFv NLDC-145 -HER2 induced HER2-specific antibodies. BALB/c mice were vaccinated with HER2, scFv NLDC-145 -neu or scFv NLDC-145 -HER2. Control animals received PBS or pcDNA3.1. Sera were collected from the vaccinated animals after two vaccinations. A HER2-specific total IgG and IgG subclass (IgG1and IgG2a) antibodies in sera from the vaccinated animals after 1:100 dilution were determined by ELISA. The mean OD405 values of pooled sera from each group (5 mice per group) were presented. Bars, SE. The background OD405 of normal mouse sera was

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Protective effects of scFv NLDC-145 -neu in transgenic BALB-neuT mice. A female BALB-neuT mice (10 mice per group) were vaccinated with neu or scFv NLDC-145 -neu in left hind limb on days -21 and -7. Control animals received pcDNA3.1 or PBS. On day 0, mice were inoculated s.c. with neu-expressing TUBO cells in opposite flank. Left panel, kinetics of tumor growth; Right panel, survival curve. *, P
    Figure Legend Snippet: Protective effects of scFv NLDC-145 -neu in transgenic BALB-neuT mice. A female BALB-neuT mice (10 mice per group) were vaccinated with neu or scFv NLDC-145 -neu in left hind limb on days -21 and -7. Control animals received pcDNA3.1 or PBS. On day 0, mice were inoculated s.c. with neu-expressing TUBO cells in opposite flank. Left panel, kinetics of tumor growth; Right panel, survival curve. *, P

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing

    scFv NLDC-145 targets tumor antigen into DCs in vivo. A schematic representation of expression vectors. scFv NLDC-145 -HER2, scFv NLDC-145 -neu, pcDNA3.1-HER2, or pcDNA3.1-neu encode under the control of a CMV promoter, all the fusion proteins consisting of an signal peptide, amino acid residues 22 to 561 of human HER2 or amino acid residues 22 to 582 of rat neu, and COOH-terminal His tag. The control plasmids pcDNA3.1-HER2 and pcDNA3.1-neu lack the NLDC-145 domains. B 293T cells grown in 100-mm dishes were transfected with various expression vectors using Lipofectamine 2000 (invitrogen). Immunoblot analysis of supernatants from 293T cells transfected with scFv NLDC-145 -HER2, scFv NLDC-145 -neu, pcDNA3.1-HER2 and pcDNA3.1-neu (lane 1, 2, 3 and 4). Vaccine proteins were probed with mouse anti-His tag mAb followed by HRP-conjugated secondary anti-mouse antibody. C left, scFv NLDC-145 -EGFP plasmid and controls as indicated were injected i.m. into mice, spleens were removed 48, 60, and 72 h thereafter, and stained with PE-conjugated anti-CD11c antibody. The GFP fluorescence in splenocytes was measured by flow cytometry. Representative results from one animal of each group 60 h post-injection. right, the expression of EGFP (MW,~25 kDa) and scFv NLDC-145 -EGFP (MW,~55 kDa) fusion protein in injected muscle tissues detected by western blotting using anti- EGFP antibody 24 h after plasmid injection with GAPDH (MW,42 kDa) as loading control. Lane 1, mice treated with pcDNA3.1 control vaccine; lane 2, mice treated with EGFP-encoding vaccine; lane 3, mice treated with scFv NLDC-145 -EGFP vaccine; D percentages of GFP + DC in total DC ( left panel ); Percentages of GFP + in CD11c-negative splenocytes ( right panel ). Bars, SE. *, P
    Figure Legend Snippet: scFv NLDC-145 targets tumor antigen into DCs in vivo. A schematic representation of expression vectors. scFv NLDC-145 -HER2, scFv NLDC-145 -neu, pcDNA3.1-HER2, or pcDNA3.1-neu encode under the control of a CMV promoter, all the fusion proteins consisting of an signal peptide, amino acid residues 22 to 561 of human HER2 or amino acid residues 22 to 582 of rat neu, and COOH-terminal His tag. The control plasmids pcDNA3.1-HER2 and pcDNA3.1-neu lack the NLDC-145 domains. B 293T cells grown in 100-mm dishes were transfected with various expression vectors using Lipofectamine 2000 (invitrogen). Immunoblot analysis of supernatants from 293T cells transfected with scFv NLDC-145 -HER2, scFv NLDC-145 -neu, pcDNA3.1-HER2 and pcDNA3.1-neu (lane 1, 2, 3 and 4). Vaccine proteins were probed with mouse anti-His tag mAb followed by HRP-conjugated secondary anti-mouse antibody. C left, scFv NLDC-145 -EGFP plasmid and controls as indicated were injected i.m. into mice, spleens were removed 48, 60, and 72 h thereafter, and stained with PE-conjugated anti-CD11c antibody. The GFP fluorescence in splenocytes was measured by flow cytometry. Representative results from one animal of each group 60 h post-injection. right, the expression of EGFP (MW,~25 kDa) and scFv NLDC-145 -EGFP (MW,~55 kDa) fusion protein in injected muscle tissues detected by western blotting using anti- EGFP antibody 24 h after plasmid injection with GAPDH (MW,42 kDa) as loading control. Lane 1, mice treated with pcDNA3.1 control vaccine; lane 2, mice treated with EGFP-encoding vaccine; lane 3, mice treated with scFv NLDC-145 -EGFP vaccine; D percentages of GFP + DC in total DC ( left panel ); Percentages of GFP + in CD11c-negative splenocytes ( right panel ). Bars, SE. *, P

    Techniques Used: In Vivo, Expressing, Transfection, Plasmid Preparation, Injection, Mouse Assay, Staining, Fluorescence, Flow Cytometry, Cytometry, Western Blot

    32) Product Images from "HBx activates FasL and mediates HepG2 cell apoptosis through MLK3-MKK7-JNKs signal module"

    Article Title: HBx activates FasL and mediates HepG2 cell apoptosis through MLK3-MKK7-JNKs signal module

    Journal: World Journal of Gastroenterology : WJG

    doi: 10.3748/wjg.v18.i13.1485

    Inhibition of HBx-induced cell apoptosis by K252a. HepG2 cells were cultured in 0.01% dimethyl sulfoxide in the absence or presence of 300 nmol/L K252a after transfection with pcDNA3.1-X and incubated for 24 h, and the cell apoptosis was examined by flow
    Figure Legend Snippet: Inhibition of HBx-induced cell apoptosis by K252a. HepG2 cells were cultured in 0.01% dimethyl sulfoxide in the absence or presence of 300 nmol/L K252a after transfection with pcDNA3.1-X and incubated for 24 h, and the cell apoptosis was examined by flow

    Techniques Used: Inhibition, Cell Culture, Transfection, Incubation, Flow Cytometry

    Apoptosis of HepG2 cells induced by hepatitis B virus X protein. HepG2 cells were cotransfected with pcDNA3.1-X and pSilencer3.1-shHBX as RNAi group, and HepG2 cells were cotransfected with pcDNA3.1-X and pSilencer3.1-H1 plasmids as negative control.
    Figure Legend Snippet: Apoptosis of HepG2 cells induced by hepatitis B virus X protein. HepG2 cells were cotransfected with pcDNA3.1-X and pSilencer3.1-shHBX as RNAi group, and HepG2 cells were cotransfected with pcDNA3.1-X and pSilencer3.1-H1 plasmids as negative control.

    Techniques Used: Negative Control

    Upregulation of Fas/FasL signaling pathway-related protein expression by hepatitis B virus X protein in a time-dependent manner. HepG2 cells were transfected with pcDNA3.1-X and incubated for various time periods as indicated. The levels of Fas, FasL
    Figure Legend Snippet: Upregulation of Fas/FasL signaling pathway-related protein expression by hepatitis B virus X protein in a time-dependent manner. HepG2 cells were transfected with pcDNA3.1-X and incubated for various time periods as indicated. The levels of Fas, FasL

    Techniques Used: Expressing, Transfection, Incubation

    Detection of hepatitis B virus X protein expression in transfected HepG2 cells. HepG2 cells were transfected with pcDNA3.1-X plasmids or cotransfected with pcDNA3.1-X and pSilencer3.1-shHBX plasmids. Forty-eight hours later, the expression of hepatitis
    Figure Legend Snippet: Detection of hepatitis B virus X protein expression in transfected HepG2 cells. HepG2 cells were transfected with pcDNA3.1-X plasmids or cotransfected with pcDNA3.1-X and pSilencer3.1-shHBX plasmids. Forty-eight hours later, the expression of hepatitis

    Techniques Used: Expressing, Transfection

    33) Product Images from "Cockayne syndrome group B protein is engaged in processing of DNA adducts of lipid peroxidation product trans-4-hydroxy-2-nonenal"

    Article Title: Cockayne syndrome group B protein is engaged in processing of DNA adducts of lipid peroxidation product trans-4-hydroxy-2-nonenal

    Journal: Mutation research

    doi: 10.1016/j.mrfmmm.2009.03.007

    Clonogenic survival of transformed CSB-deficient, CS1AN/pc3.1, (○) and CSB-proficient, CS1AN/pc3.1-CSBwt (●) cell lines after 2 h of treatment with HNE. HNE survival curve represents the mean of three independent experiments. Error bars
    Figure Legend Snippet: Clonogenic survival of transformed CSB-deficient, CS1AN/pc3.1, (○) and CSB-proficient, CS1AN/pc3.1-CSBwt (●) cell lines after 2 h of treatment with HNE. HNE survival curve represents the mean of three independent experiments. Error bars

    Techniques Used: Transformation Assay

    Sister chromatid exchanges (SCEs) induced by HNE in CSB-deficient, CS1AN/pc3.1 (black bars) and CSB-proficient, CS1AN/pc3.1-CSBwt (white bars) cell lines. Error bars represent standard errors of the mean. Statistically significant differences are indicated
    Figure Legend Snippet: Sister chromatid exchanges (SCEs) induced by HNE in CSB-deficient, CS1AN/pc3.1 (black bars) and CSB-proficient, CS1AN/pc3.1-CSBwt (white bars) cell lines. Error bars represent standard errors of the mean. Statistically significant differences are indicated

    Techniques Used:

    34) Product Images from "UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras"

    Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1215-4

    UBIAD1 inhibits the Ras/ERK signaling pathway. a UBIAD1 reduced cell viability in T24 bladder cancer cells. T24 cells were transiently transfected with pcDNA3.1-UBIAD1 plasmid. Twenty-four hours after transfection, cell viability was detected by the MTT assay. *** p
    Figure Legend Snippet: UBIAD1 inhibits the Ras/ERK signaling pathway. a UBIAD1 reduced cell viability in T24 bladder cancer cells. T24 cells were transiently transfected with pcDNA3.1-UBIAD1 plasmid. Twenty-four hours after transfection, cell viability was detected by the MTT assay. *** p

    Techniques Used: Transfection, Plasmid Preparation, MTT Assay

    35) Product Images from "DNA Vaccine Encoding Middle East Respiratory Syndrome Coronavirus S1 Protein Induces Protective Immune Responses in Mice"

    Article Title: DNA Vaccine Encoding Middle East Respiratory Syndrome Coronavirus S1 Protein Induces Protective Immune Responses in Mice

    Journal: Vaccine

    doi: 10.1016/j.vaccine.2017.02.063

    DNA vaccine-induced antigen-specific cellular immune responses Splenocytes were isolated two weeks following the second immunization and stimulated with or without the purified RBD protein. The S1-specific IFN-γ and IL-4 activities in splenocytes were evaluated using commercial ELISpot kits. SFCs secreting IL-4 ( A ) and IFN-γ ( B ) were enumerated in an automated ELISpot reader. The ability of the pcDNA3.1-S1 DNA vaccine to induce IFN-γ- and IL-4-expression in antigen-specific CD4 + and CD8 + T cells was analyzed by intracellular cytokine staining. Cells were stained with combined mouse anti-CD4-FITC and anti-CD8-PE, anti-IFN-γ-PE-Cy7 and anti-IL-4-PE-Cy3 monoclonal antibodies. CD4 + T cells expressing IFN-γ ( C ) and IL-4 ( D ) and CD8 + T cells expressing IFN-γ ( E ) and IL-4 ( F ) were analyzed in a FACSAria ™ Cell Sorter. n= 3 mice/group/time point. Data are shown as the means ± SDs and were analyzed by unpaired Student’s t test. (* P
    Figure Legend Snippet: DNA vaccine-induced antigen-specific cellular immune responses Splenocytes were isolated two weeks following the second immunization and stimulated with or without the purified RBD protein. The S1-specific IFN-γ and IL-4 activities in splenocytes were evaluated using commercial ELISpot kits. SFCs secreting IL-4 ( A ) and IFN-γ ( B ) were enumerated in an automated ELISpot reader. The ability of the pcDNA3.1-S1 DNA vaccine to induce IFN-γ- and IL-4-expression in antigen-specific CD4 + and CD8 + T cells was analyzed by intracellular cytokine staining. Cells were stained with combined mouse anti-CD4-FITC and anti-CD8-PE, anti-IFN-γ-PE-Cy7 and anti-IL-4-PE-Cy3 monoclonal antibodies. CD4 + T cells expressing IFN-γ ( C ) and IL-4 ( D ) and CD8 + T cells expressing IFN-γ ( E ) and IL-4 ( F ) were analyzed in a FACSAria ™ Cell Sorter. n= 3 mice/group/time point. Data are shown as the means ± SDs and were analyzed by unpaired Student’s t test. (* P

    Techniques Used: Isolation, Purification, Enzyme-linked Immunospot, Expressing, Staining, Mouse Assay

    DNA vaccine-induced neutralizing antibody against MERS-CoV Serum samples were collected by retro-orbital plexus puncture at weeks 1, 2, 4, 5, 7 and 8. Anti-MERS-S antibody levels in serum were assessed by indirect ELISA with the purified RBD protein as the detection antigen, and shown as end-point dilution titers. The horizontal dotted line indicates limit of determination (LOD). n= 6 mice/group/time point. The ELISA titers of serum samples from pcDNA3.1-S, pcDNA3.1-SΔCD, pcDNA3.1-S1 at weeks 2, 5 and 8. Data are shown as the means ± SDs and were analyzed by one-way ANOVA. (**** P
    Figure Legend Snippet: DNA vaccine-induced neutralizing antibody against MERS-CoV Serum samples were collected by retro-orbital plexus puncture at weeks 1, 2, 4, 5, 7 and 8. Anti-MERS-S antibody levels in serum were assessed by indirect ELISA with the purified RBD protein as the detection antigen, and shown as end-point dilution titers. The horizontal dotted line indicates limit of determination (LOD). n= 6 mice/group/time point. The ELISA titers of serum samples from pcDNA3.1-S, pcDNA3.1-SΔCD, pcDNA3.1-S1 at weeks 2, 5 and 8. Data are shown as the means ± SDs and were analyzed by one-way ANOVA. (**** P

    Techniques Used: Indirect ELISA, Purification, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Construction and verification of DNA vaccine Schematic diagrams of the construction of DNA vaccines encoding different fragments of MERS-CoV spike protein ( A ). Western blot analyses of MERS-CoV spike protein expression in vitro . Lysates from pcDNA3.1-S, pcDNA3.1-SΔCD, pcDNA3.1-S1 transfected 293T cells (lane 1–3) and lysates from pcDNA3.1-Empty transfected 293T cells (lane 4) were incubated with mouse anti-MERS-S1 monoclonal antibodies and mouse anti-β-tubulin monoclonal antibodies ( B ). The schematic of the experiment ( C ).
    Figure Legend Snippet: Construction and verification of DNA vaccine Schematic diagrams of the construction of DNA vaccines encoding different fragments of MERS-CoV spike protein ( A ). Western blot analyses of MERS-CoV spike protein expression in vitro . Lysates from pcDNA3.1-S, pcDNA3.1-SΔCD, pcDNA3.1-S1 transfected 293T cells (lane 1–3) and lysates from pcDNA3.1-Empty transfected 293T cells (lane 4) were incubated with mouse anti-MERS-S1 monoclonal antibodies and mouse anti-β-tubulin monoclonal antibodies ( B ). The schematic of the experiment ( C ).

    Techniques Used: Western Blot, Expressing, In Vitro, Transfection, Incubation

    36) Product Images from "Differential requirement for the ATPase domain of the Cockayne syndrome group B gene in the processing of UV-induced DNA damage and 8-oxoguanine lesions in human cells"

    Article Title: Differential requirement for the ATPase domain of the Cockayne syndrome group B gene in the processing of UV-induced DNA damage and 8-oxoguanine lesions in human cells

    Journal: Nucleic Acids Research

    doi:

    Ratios of gene expression from the NIA arrays comparing the following cell lines: CS1AN/pc3.1-CSBwt versus CS1AN/pc3.1 ( A ), CS1AN/pc3.1-CSBwt versus CS1AN/pc3.1-CSBE646Q ( B ) and CS1AN/pc3.1 versus CS1AN/pc3.1-CSBE646Q ( C ). The two outlying genes in (A) and (B) are the same and are identified as the human guanine nucleotide exchange factor DBS and human mRNA for KIAAA232 gene, whose function is unknown.
    Figure Legend Snippet: Ratios of gene expression from the NIA arrays comparing the following cell lines: CS1AN/pc3.1-CSBwt versus CS1AN/pc3.1 ( A ), CS1AN/pc3.1-CSBwt versus CS1AN/pc3.1-CSBE646Q ( B ) and CS1AN/pc3.1 versus CS1AN/pc3.1-CSBE646Q ( C ). The two outlying genes in (A) and (B) are the same and are identified as the human guanine nucleotide exchange factor DBS and human mRNA for KIAAA232 gene, whose function is unknown.

    Techniques Used: Expressing

    Incision of 8-oxoguanine-containing and thymine glycol-containing oligonucleotides by whole-cell extracts. ( A ) An 8-oxoguanine-containing and a thymine glycol-containing 5′-end-labeled oligonucleotide duplex were incubated in the same reaction mixture with 20 µg of protein from whole-cell extracts. Reactions were incubated for 6 h and terminated by addition of SDS and proteinase K. The products of incision were separated from the substrates on a 20% polyacrylamide gel. Following incision at the lesion, the 8-oxoguanine-containing oligonucleotide is cut down to a 10mer and the thymine glycol-containing oligonucleotide is cut down to a 15mer (lane 1, marker; lane 2, CS1AN/pc3.1-CSBwt; lane 3, CS1AN/pc3.1-CSBE646Q; lane 4, CS1AN/pc3.1). ( B ) The gels were quantified with a PhosphorImager (ImageQuant) and the percent incision was calculated.
    Figure Legend Snippet: Incision of 8-oxoguanine-containing and thymine glycol-containing oligonucleotides by whole-cell extracts. ( A ) An 8-oxoguanine-containing and a thymine glycol-containing 5′-end-labeled oligonucleotide duplex were incubated in the same reaction mixture with 20 µg of protein from whole-cell extracts. Reactions were incubated for 6 h and terminated by addition of SDS and proteinase K. The products of incision were separated from the substrates on a 20% polyacrylamide gel. Following incision at the lesion, the 8-oxoguanine-containing oligonucleotide is cut down to a 10mer and the thymine glycol-containing oligonucleotide is cut down to a 15mer (lane 1, marker; lane 2, CS1AN/pc3.1-CSBwt; lane 3, CS1AN/pc3.1-CSBE646Q; lane 4, CS1AN/pc3.1). ( B ) The gels were quantified with a PhosphorImager (ImageQuant) and the percent incision was calculated.

    Techniques Used: Labeling, Incubation, Marker

    Incision of 8-oxoguanine (8-oxodG)-containing and 5-hydroxyl-dCytosine (5-OHdC)-containing oligonucleotides by whole-cell extracts (C, no extract control; Wt, CS1AN/pc3.1-CSBwt; M, CS1AN/pc3.1-CSBE646Q ATPase mutant; V, CS1AN/pc3.1 vector control). ( A ) Oligonucleotides employed. ( B ) The 8-oxodeoxyguanine-containing 5′-labeled oligonucleotide duplex and a 5-hydroxyl-dCytosine-containing 5′-labeled oligonucleotide duplex were incubated in the reaction mixture with 20 µg of protein from whole-cell extracts. Reactions were incubated for 6 h for 5-hydroxyl-dCytosine and the hours indicated for 8-oxodG at 37°C and terminated by addition of SDS and proteinase K. The products of incision were separated from the substrates on a 20% polyacrylamide gel. Following incision at the lesion, both oligonucleotides are cut down to a 10mer. The gels were quantified with a PhosphorImager (ImageQuant). ( C ) The percentage of 5-hydroxyl-dCytosine incision was calculated for the 6 h time point. ( D ) Incision kinetics of 8-oxoguanine were calculated, extracts were analyzed in triplicate from two biological extracts.
    Figure Legend Snippet: Incision of 8-oxoguanine (8-oxodG)-containing and 5-hydroxyl-dCytosine (5-OHdC)-containing oligonucleotides by whole-cell extracts (C, no extract control; Wt, CS1AN/pc3.1-CSBwt; M, CS1AN/pc3.1-CSBE646Q ATPase mutant; V, CS1AN/pc3.1 vector control). ( A ) Oligonucleotides employed. ( B ) The 8-oxodeoxyguanine-containing 5′-labeled oligonucleotide duplex and a 5-hydroxyl-dCytosine-containing 5′-labeled oligonucleotide duplex were incubated in the reaction mixture with 20 µg of protein from whole-cell extracts. Reactions were incubated for 6 h for 5-hydroxyl-dCytosine and the hours indicated for 8-oxodG at 37°C and terminated by addition of SDS and proteinase K. The products of incision were separated from the substrates on a 20% polyacrylamide gel. Following incision at the lesion, both oligonucleotides are cut down to a 10mer. The gels were quantified with a PhosphorImager (ImageQuant). ( C ) The percentage of 5-hydroxyl-dCytosine incision was calculated for the 6 h time point. ( D ) Incision kinetics of 8-oxoguanine were calculated, extracts were analyzed in triplicate from two biological extracts.

    Techniques Used: Mutagenesis, Plasmid Preparation, Labeling, Incubation

    Western blot analysis of whole-cell extracts from CS1AN cell lines transfected with the vector pc3.1 or pc3.1 containing the wild-type human CSB gene (pc3.1-CSBwt) or CSB containing a mutation in the ATPase domain II (pC3.1-CSBE646Q). Lane 1, CS1AN/pc3.1-CSBwt; lane 2, CS1AN/pc3.1-CSBE646Q; lane 3, CS1AN/pc3.1. Blots were probed with a human anti-ERCC6 IgG fraction monoclonal antibody. Arrow indicates the full-length 168 kDa CSB protein. Purified CSB protein was run as a control.
    Figure Legend Snippet: Western blot analysis of whole-cell extracts from CS1AN cell lines transfected with the vector pc3.1 or pc3.1 containing the wild-type human CSB gene (pc3.1-CSBwt) or CSB containing a mutation in the ATPase domain II (pC3.1-CSBE646Q). Lane 1, CS1AN/pc3.1-CSBwt; lane 2, CS1AN/pc3.1-CSBE646Q; lane 3, CS1AN/pc3.1. Blots were probed with a human anti-ERCC6 IgG fraction monoclonal antibody. Arrow indicates the full-length 168 kDa CSB protein. Purified CSB protein was run as a control.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Purification

    37) Product Images from "TAK1 inhibition attenuates both inflammation and fibrosis in experimental pneumoconiosis"

    Article Title: TAK1 inhibition attenuates both inflammation and fibrosis in experimental pneumoconiosis

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2017.23

    Validation of TAK1 as a molecular target of resveratrol and identification of key residues in TAK1. ( a ) Immunoprecipitation using anti-resveratrol antibody to examine the interaction between TAK1 and resveratrol (Res) in alveolar macrophage NR8383 (left) and lung fibroblasts WI-38 cells (right). TAK1 in the immunoprecipitate and lysate was examined by western blotting (upper: representative images; bottom: relative bands intensity). ( b ) Reference standard of resveratrol (left upper) and immunoprecipitation using anti-TAK1 antibody to examine the interaction between resveratrol and TAK1 in NR8383 (left bottom) and WI-38 cells (right). Resveratrol in the immunoprecipitate was examined by LC-MS/MS. There were two peaks for trans - and cis -resveratrol according to the reference standard. ( c ) Close-up view showing key residues on TAK1 in two predicted binding conformations between TAK1 and resveratrol in molecule docking. ( d ) Immunoprecipitation using anti-resveratrol antibody to examine the interaction between Flag-TAK1 wild-type or Flag-TAK1 mutants (N161R, A107R and N161R/A107R) and resveratrol in NR8383 (left) and WI-38 cells (right). The cells were transfected with pcDNA3.1-based expression vectors for Flag-TAK1 wild-type or Flag-TAK1 mutants (N161R, A107R and N161R/A107R). Flag-TAK1 or Flag-TAK1 mutants in immunoprecipitates and lysates was determined by western blotting (upper: representative images; bottom: relative bands intensity). ( e ) Immunoprecipitation using anti-Flag antibody to examine the interaction between resveratrol and Flag-TAK1 wild-type or Flag-TAK1 mutants in NR8383 cells (upper) and WI-38 cells (bottom). Resveratrol in immunoprecipitate was examined by LC-MS/MS. Experiments were performed at least three times. Res, resveratrol; ND, not detectable.
    Figure Legend Snippet: Validation of TAK1 as a molecular target of resveratrol and identification of key residues in TAK1. ( a ) Immunoprecipitation using anti-resveratrol antibody to examine the interaction between TAK1 and resveratrol (Res) in alveolar macrophage NR8383 (left) and lung fibroblasts WI-38 cells (right). TAK1 in the immunoprecipitate and lysate was examined by western blotting (upper: representative images; bottom: relative bands intensity). ( b ) Reference standard of resveratrol (left upper) and immunoprecipitation using anti-TAK1 antibody to examine the interaction between resveratrol and TAK1 in NR8383 (left bottom) and WI-38 cells (right). Resveratrol in the immunoprecipitate was examined by LC-MS/MS. There were two peaks for trans - and cis -resveratrol according to the reference standard. ( c ) Close-up view showing key residues on TAK1 in two predicted binding conformations between TAK1 and resveratrol in molecule docking. ( d ) Immunoprecipitation using anti-resveratrol antibody to examine the interaction between Flag-TAK1 wild-type or Flag-TAK1 mutants (N161R, A107R and N161R/A107R) and resveratrol in NR8383 (left) and WI-38 cells (right). The cells were transfected with pcDNA3.1-based expression vectors for Flag-TAK1 wild-type or Flag-TAK1 mutants (N161R, A107R and N161R/A107R). Flag-TAK1 or Flag-TAK1 mutants in immunoprecipitates and lysates was determined by western blotting (upper: representative images; bottom: relative bands intensity). ( e ) Immunoprecipitation using anti-Flag antibody to examine the interaction between resveratrol and Flag-TAK1 wild-type or Flag-TAK1 mutants in NR8383 cells (upper) and WI-38 cells (bottom). Resveratrol in immunoprecipitate was examined by LC-MS/MS. Experiments were performed at least three times. Res, resveratrol; ND, not detectable.

    Techniques Used: Immunoprecipitation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Binding Assay, Transfection, Expressing

    38) Product Images from "TWEAK Negatively Regulates Human Dicer"

    Article Title: TWEAK Negatively Regulates Human Dicer

    Journal: Non-Coding RNA

    doi: 10.3390/ncrna2040012

    TWEAK dose-dependently reduces pre-microRNA conversion into mature microRNA by Dicer. ( a , b ) Cleared lysates (50 µg of proteins) prepared from HEK 293 cells, transfected with increasing amounts of pcDNA3.1-Flag-TWEAK vector (0–20 µg of DNA), were incubated with 5′ 32 P-labeled human pre-miR-223 (miRBase acc. no. MI0000300) or pre-let-7c (miRBase acc. no. MI0018703). ( a ) The 32 P-labeled pre-microRNA substrates and cleavage products of Dicer were revealed by denaturing polyacrylamide gel electrophoresis (PAGE)/autoradiography; ( b ) The ≈20-nt bands corresponding to the mature microRNA products were analyzed by densitometry using the ImageJ ® software; ( c ) The Dicer protein level in the HEK 293 cell lysates was analyzed by immunoblotting using anti-Dicer antibody, with anti-tubulin as a reference.
    Figure Legend Snippet: TWEAK dose-dependently reduces pre-microRNA conversion into mature microRNA by Dicer. ( a , b ) Cleared lysates (50 µg of proteins) prepared from HEK 293 cells, transfected with increasing amounts of pcDNA3.1-Flag-TWEAK vector (0–20 µg of DNA), were incubated with 5′ 32 P-labeled human pre-miR-223 (miRBase acc. no. MI0000300) or pre-let-7c (miRBase acc. no. MI0018703). ( a ) The 32 P-labeled pre-microRNA substrates and cleavage products of Dicer were revealed by denaturing polyacrylamide gel electrophoresis (PAGE)/autoradiography; ( b ) The ≈20-nt bands corresponding to the mature microRNA products were analyzed by densitometry using the ImageJ ® software; ( c ) The Dicer protein level in the HEK 293 cell lysates was analyzed by immunoblotting using anti-Dicer antibody, with anti-tubulin as a reference.

    Techniques Used: Transfection, Plasmid Preparation, Incubation, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Software

    TWEAK impairs microRNA-guided RNA silencing of a reporter gene induced by a pre-microRNA. HEK 293 cells expressing Flag-TWEAK (or transfected with an empty pcDNA3.1-Flag vector; Flag) were transfected with the silencing inducer pre-miR-328a (or a negative pre-microRNA control targeting a deleted region in Rluc ; shNEG), and a Rluc reporter gene [ 16 ] coupled with three copies of a natural miR-328a binding site in the 3′ untranslated region (UTR) of the Rluc reporter gene. Cells were harvested 18 h later for the successive measurements of Rluc and Fluc activities ( n = 3 experiments, in duplicate). * p
    Figure Legend Snippet: TWEAK impairs microRNA-guided RNA silencing of a reporter gene induced by a pre-microRNA. HEK 293 cells expressing Flag-TWEAK (or transfected with an empty pcDNA3.1-Flag vector; Flag) were transfected with the silencing inducer pre-miR-328a (or a negative pre-microRNA control targeting a deleted region in Rluc ; shNEG), and a Rluc reporter gene [ 16 ] coupled with three copies of a natural miR-328a binding site in the 3′ untranslated region (UTR) of the Rluc reporter gene. Cells were harvested 18 h later for the successive measurements of Rluc and Fluc activities ( n = 3 experiments, in duplicate). * p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Binding Assay

    Dicer and TWEAK proteins form a complex likely independent of RNA in HEK 293 cells. HEK 293 cells were transiently transfected with pcDNA3.1-Flag-TWEAK vector, or an empty pcDNA3.1-Flag vector (Flag), prior to Flag-TWEAK protein immunoprecipitation (IP) from untreated lysate (lanes 1–3 and 6–8) or lysate treated with RNases A/T1/V1 (+RNases; lanes 4–5 and 9–10). ( a ) The presence of Flag-TWEAK and Dicer proteins in the lysates (Input) and the IPs was verified by immunoblot (IB) analysis using anti-Flag and anti-Dicer antibodies. The two bands immunoreactive to the anti-Flag antibody likely correspond to different posttranslational forms of the Flag-tagged TWEAK protein. The band across lanes 6 to 10 of the IB anti-Flag panel likely corresponds to the light chain of mouse IgGs; ( b ) RNA degradation upon treatment with RNases A/T1/V1 was confirmed by agarose gel electrophoresis and ethidium bromide staining.
    Figure Legend Snippet: Dicer and TWEAK proteins form a complex likely independent of RNA in HEK 293 cells. HEK 293 cells were transiently transfected with pcDNA3.1-Flag-TWEAK vector, or an empty pcDNA3.1-Flag vector (Flag), prior to Flag-TWEAK protein immunoprecipitation (IP) from untreated lysate (lanes 1–3 and 6–8) or lysate treated with RNases A/T1/V1 (+RNases; lanes 4–5 and 9–10). ( a ) The presence of Flag-TWEAK and Dicer proteins in the lysates (Input) and the IPs was verified by immunoblot (IB) analysis using anti-Flag and anti-Dicer antibodies. The two bands immunoreactive to the anti-Flag antibody likely correspond to different posttranslational forms of the Flag-tagged TWEAK protein. The band across lanes 6 to 10 of the IB anti-Flag panel likely corresponds to the light chain of mouse IgGs; ( b ) RNA degradation upon treatment with RNases A/T1/V1 was confirmed by agarose gel electrophoresis and ethidium bromide staining.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Agarose Gel Electrophoresis, Staining

    39) Product Images from "PDX1 homeobox protein expression in pseudopyloric glands and gastric carcinomas"

    Article Title: PDX1 homeobox protein expression in pseudopyloric glands and gastric carcinomas

    Journal: Gut

    doi: 10.1136/gut.2003.026609

    Confirmation of anti-pancreatic-duodenal homeobox 1 (PDX1) antibody by western blot analysis. (A) Lane 1: Band was not detected in GT3TKB transfected with pcDNA3.1-PDX1, reacted with pre-immunised serum; lane 2: no band was seen in GT3TKB transfected with mammalian expression vector without PDX1 cDNA, reacted with anti-PDX1 antibody; lane 3: a predicted band was detected in GT3TKB transfected with pcDNA3.1-PDX1, reacted with anti-PDX1 antibody; lanes 4, 5: the band disappeared in GT3TKB transfected with pcDNA3.1-PDX1, reacted with anti-PDX1 antibody treated with 10 and 50 µg of glutathione-S-transferase (GST)-PDX1, respectively; lanes 6, 7: the band did not fade out in GT3TKB transfected with pcDNA3.1-PDX1, reacted with anti-PDX1 antibody treated with 10 and 50 µg of GST, respectively. α-Tubulin was analysed as an internal control. (B) Lane 1: a band appears in extracted proteins from the normal antrum; lanes 2, 3: no band was observed in proteins from the normal corpus and colon, respectively.
    Figure Legend Snippet: Confirmation of anti-pancreatic-duodenal homeobox 1 (PDX1) antibody by western blot analysis. (A) Lane 1: Band was not detected in GT3TKB transfected with pcDNA3.1-PDX1, reacted with pre-immunised serum; lane 2: no band was seen in GT3TKB transfected with mammalian expression vector without PDX1 cDNA, reacted with anti-PDX1 antibody; lane 3: a predicted band was detected in GT3TKB transfected with pcDNA3.1-PDX1, reacted with anti-PDX1 antibody; lanes 4, 5: the band disappeared in GT3TKB transfected with pcDNA3.1-PDX1, reacted with anti-PDX1 antibody treated with 10 and 50 µg of glutathione-S-transferase (GST)-PDX1, respectively; lanes 6, 7: the band did not fade out in GT3TKB transfected with pcDNA3.1-PDX1, reacted with anti-PDX1 antibody treated with 10 and 50 µg of GST, respectively. α-Tubulin was analysed as an internal control. (B) Lane 1: a band appears in extracted proteins from the normal antrum; lanes 2, 3: no band was observed in proteins from the normal corpus and colon, respectively.

    Techniques Used: Western Blot, Transfection, Expressing, Plasmid Preparation

    40) Product Images from "Functional Properties of a New Voltage-dependent Calcium Channel α2δ Auxiliary Subunit Gene (CACNA2D2) *"

    Article Title: Functional Properties of a New Voltage-dependent Calcium Channel α2δ Auxiliary Subunit Gene (CACNA2D2) *

    Journal: The Journal of biological chemistry

    doi:

    A , in vitro transcription and translation of α 2 δ -2. Lane 1 , in vitro transcription and translation of α 2 δ -2 in expression vector pcDNA3.1. Lane 2 , no DNA was added in the same reaction. The arrow indicates the expected 130 kDa product. B , Western blot analysis of transfection of NCI-H1299 cells with α 2 δ -2. Lane 1 , transfection of NCI-H1299 cells with α 2 δ -2 in expression vector pcDNA3.1. Lane 2 , transfection of NCI-H1299 cells with pcDNA3.1 vector alone. Affinity-purified anti- α 2 δ -2 peptide antibody was used to detect the protein product. The arrow indicates the expected protein product. Sizes of the prestained protein molecular weight markers are indicated on the right. C , 40 μ g of protein from tumor cell lysates were loaded in each lane. Lane 1 , NCI-H2O77 (adenocarcinoma); lane 2 , NCI-H358 (adenocarcinoma); lane 3 , NCI-H2106 (large cell neuroendocrine carcinoma); lane 4 , NCI-H1299 (large cell carcinoma) cells.
    Figure Legend Snippet: A , in vitro transcription and translation of α 2 δ -2. Lane 1 , in vitro transcription and translation of α 2 δ -2 in expression vector pcDNA3.1. Lane 2 , no DNA was added in the same reaction. The arrow indicates the expected 130 kDa product. B , Western blot analysis of transfection of NCI-H1299 cells with α 2 δ -2. Lane 1 , transfection of NCI-H1299 cells with α 2 δ -2 in expression vector pcDNA3.1. Lane 2 , transfection of NCI-H1299 cells with pcDNA3.1 vector alone. Affinity-purified anti- α 2 δ -2 peptide antibody was used to detect the protein product. The arrow indicates the expected protein product. Sizes of the prestained protein molecular weight markers are indicated on the right. C , 40 μ g of protein from tumor cell lysates were loaded in each lane. Lane 1 , NCI-H2O77 (adenocarcinoma); lane 2 , NCI-H358 (adenocarcinoma); lane 3 , NCI-H2106 (large cell neuroendocrine carcinoma); lane 4 , NCI-H1299 (large cell carcinoma) cells.

    Techniques Used: In Vitro, Expressing, Plasmid Preparation, Western Blot, Transfection, Affinity Purification, Molecular Weight

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    Sequencing:

    Article Title: MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo
    Article Snippet: .. The correct sequences of amplified fragment were verified by sequencing, double digested with HindIII and BamHI, and cloned into pcDNA3.1 vector (Invitrogen), carrying neomycin resistance gene. .. Transfection was performed using the Lipofectamine™ 2000 transfection reagent (invitrogen) according to the manufacturer's instructions.

    Plasmid Preparation:

    Article Title: Identification and characterization of novel PAX8 mutations in Congenital Hypothyroidism(CH) in a Chinese population
    Article Snippet: .. PCR products were cloned into the pCDNA3.1 expression vector (Invitrogen, Carlsbad, CA, USA) using KpnI and XbaI restriction sites introduced into the primers to obtain the wild-type (WT) PAX8-pCDNA3.1( PAX8WT-pCDNA3.1). .. Mutations were introduced using the Quick Change Mutagenesis kit (Transgene Biotech, Beijing, China), following the manufacturer's protocol, and the following primers: PAX8 D94N-pCDNA3.1 construct, 5′-GTGGAGAAGATTGGGAACTACAAACG-3′ and 5′-TCCCAATCTTCTCCACCACCTTGGGG-3′; and PAX8 G41V-pCDNA3.1 construct, 5′-ACCTGGCCCACCAGGTTGTAAGGCCC-3′ and 5′-ACCTGGTGGGCCAGGTCTACGATGCG-3′.

    Article Title: MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo
    Article Snippet: .. The correct sequences of amplified fragment were verified by sequencing, double digested with HindIII and BamHI, and cloned into pcDNA3.1 vector (Invitrogen), carrying neomycin resistance gene. .. Transfection was performed using the Lipofectamine™ 2000 transfection reagent (invitrogen) according to the manufacturer's instructions.

    Article Title: Inhibition of mitochondrial respiration by nitric oxide rapidly stimulates cytoprotective GLUT3-mediated glucose uptake through 5?-AMP-activated protein kinase
    Article Snippet: .. Full-length GLUT3 or GLUT1 cDNAs, digested from pBS-rG3, were also subcloned into the Eco RI site of the pcDNA3 mammalian expression vector (Invitrogen, Madrid, Spain; pcDNA3-G3). ..

    Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras
    Article Snippet: .. Enhanced green fluorescent protein (EGFP) vectors pEGFP-N1, pEGFP-C1, pCasper3-BG (TAGBFP-GFP), mammalian expression vector pcDNA3.1 and TAGBFP were obtained from Invitrogen. pDsRed-Golgi vector was obtained from Clontech, previously described . .. For construction of DsRed-H-Ras, full-length human H-Ras cDNA was amplified by PCR and cloned with DsRed-Monomer into the pcDNA3.1 vector.

    Article Title: Transient mismatch repair gene transfection for functional analysis of genetic hMLH1 and hMSH2 variants
    Article Snippet: .. The complete wild type cDNA for hMSH2 was subcloned from laboratory isolates into the pcDNA3.1+ vector (Invitrogen, Groningen, Netherlands), placing the cDNA under the control of the CMV promoter. .. Inserts for hMSH2 wt cDNA were amplified using primers bearing BamHI (sense) or XhoI (antisense) restriction sites and ligated in the appropriate sites of the vector.

    Article Title: CCN1 contributes to skin connective tissue aging by inducing age-associated secretory phenotype in human skin dermal fibroblasts
    Article Snippet: .. For transfection, human CCN1 cDNA expression vector was cloned into pCDNA3.1 expression vector (Invitrogen, Carlsbad, CA), as described previously (Quan et al. ). .. Human skin dermal fibroblasts were transiently transfected by electroporation (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer’s protocol.

    Article Title: A viral gene that activates lytic cycle expression of Kaposi's sarcoma-associated herpesvirus
    Article Snippet: .. These PCR fragments were cloned into the pcDNA3.1 expression vector (Invitrogen) and designated as KSHV/gRta. .. Primer A and primer B were used to generate PCR products from total BC-1 DNA in a separate reaction.

    Article Title: GPC3 reduces cell proliferation in renal carcinoma cell lines
    Article Snippet: .. Transfection The pcDNA3.1/GPC3 expression vector and pcDNA3.1 (empty vector) were transfected into ACHN and 786-O cell lines using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual. .. RNA extraction and qRT-PCR Total RNA was extracted using TRIzol reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions.

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    Thermo Fisher pcdna3 1
    SOCS1 physically interacts with RhTRIM5α. HEK293T cells were co-transfected with 1.0 µg of pRhTRIM5α-HA and 2.0 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 3.0 µg per sample with <t>pcDNA3.1.</t> Two days post-transfection, whole cell lysates were subjected to immunoprecipitation for RhTRIM5α with anti-HA antibody. Input lysates (left panels) and precipitated proteins (right panels) were separated by SDS-PAGE and proteins were detected by immunoblot analyses with anti-SOCS1 (upper panels) and anti-HA (RhTRIM5α-HA, lower panels) antibodies.
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    SOCS1 physically interacts with RhTRIM5α. HEK293T cells were co-transfected with 1.0 µg of pRhTRIM5α-HA and 2.0 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 3.0 µg per sample with pcDNA3.1. Two days post-transfection, whole cell lysates were subjected to immunoprecipitation for RhTRIM5α with anti-HA antibody. Input lysates (left panels) and precipitated proteins (right panels) were separated by SDS-PAGE and proteins were detected by immunoblot analyses with anti-SOCS1 (upper panels) and anti-HA (RhTRIM5α-HA, lower panels) antibodies.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: SOCS1 physically interacts with RhTRIM5α. HEK293T cells were co-transfected with 1.0 µg of pRhTRIM5α-HA and 2.0 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 3.0 µg per sample with pcDNA3.1. Two days post-transfection, whole cell lysates were subjected to immunoprecipitation for RhTRIM5α with anti-HA antibody. Input lysates (left panels) and precipitated proteins (right panels) were separated by SDS-PAGE and proteins were detected by immunoblot analyses with anti-SOCS1 (upper panels) and anti-HA (RhTRIM5α-HA, lower panels) antibodies.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Transfection, Immunoprecipitation, SDS Page

    SOCS1 is detected in purified HIV-1 VLPs. HEK293T cells were co-transfected with 2.4 µg of pNL4-3, 2.4 µg of pRhTRIM5α-HA and 2.4 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1. (A) Relative viral titer in the supernatants was analyzed with TZM-bl indicator cells two days post-transfection. The titer of virus produced in the absence of RhTRIM5α and SOCS1 (Ct) was arbitrarily set as 100%. S1, T5α or T5α/S1 indicate virions produced in the presence of SOCS1 alone, RhTRIM5α-HA alone or both RhTRIM5α-HA and SOCS1, respectively. Producer cell lysates (B, left panels) and purified HIV-1 VLPs purified through a 20% sucrose layer (B, right panels) were subjected to immunoblot analysis. Proteins were detected with anti-HA (RhTRIM5α-HA, top panels), anti-SOCS1 (middle panels) and anti-HIV-1 p24 (bottom panels) antibodies.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: SOCS1 is detected in purified HIV-1 VLPs. HEK293T cells were co-transfected with 2.4 µg of pNL4-3, 2.4 µg of pRhTRIM5α-HA and 2.4 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1. (A) Relative viral titer in the supernatants was analyzed with TZM-bl indicator cells two days post-transfection. The titer of virus produced in the absence of RhTRIM5α and SOCS1 (Ct) was arbitrarily set as 100%. S1, T5α or T5α/S1 indicate virions produced in the presence of SOCS1 alone, RhTRIM5α-HA alone or both RhTRIM5α-HA and SOCS1, respectively. Producer cell lysates (B, left panels) and purified HIV-1 VLPs purified through a 20% sucrose layer (B, right panels) were subjected to immunoblot analysis. Proteins were detected with anti-HA (RhTRIM5α-HA, top panels), anti-SOCS1 (middle panels) and anti-HIV-1 p24 (bottom panels) antibodies.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Purification, Transfection, Produced

    SOCS1 affects RhTRIM5α expression in a dose-dependent manner. HEK293T cells were co-transfected with 0.1 µg of pNL4-3 or pUC18 with increasing amounts of pHuSOCS1 (0, 0.0375, 0.075, 0.15, 0.3 and 0.6 µg) and with or without 0.3 µg of pRhTRIM5α-HA. The amount of plasmid DNA was adjusted to 1.0 µg with pcDNA3.1. Two days post-transfection, viral titer in the culture supernatants was analyzed with TZM-bl indicator cells. (A) Relative viral titer obtained without exogenous SOCS1 and RhTRIM5α was arbitrarily set as 100%. The results are shown as an average of three independent experiments with standard deviation. (B) Whole cell lysates in panel (A) were subjected to immunoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (C) Relative band intensities of RhTRIM5α and SOCS1 in panel (B) were normalized with that of GAPDH. The normalized band intensity of RhTRIM5α (upper panels) in the absence of SOCS1 and that of SOCS1 (lower panels) without exogenous SOCS1 were arbitrarily set as 100%. (D) Effect of SOCS1 expression on transcriptional activity. HEK293T cell were co-transfected with 0.05 µg of pGL4.84-EF1α-hRlucCP (EF1α) or pcDNA3.1-Luc (CMV) together with 0.45 µg of pcDNA3.1 or pHuSOCS1. The amount of plasmid DNA was adjusted to 0.5 µg with pcDNA3.1. Two days after transfection, luciferase activity was determined and normalized with protein concentration (Luc/protein). The results are shown as an average of three independent experiments with standard deviation.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: SOCS1 affects RhTRIM5α expression in a dose-dependent manner. HEK293T cells were co-transfected with 0.1 µg of pNL4-3 or pUC18 with increasing amounts of pHuSOCS1 (0, 0.0375, 0.075, 0.15, 0.3 and 0.6 µg) and with or without 0.3 µg of pRhTRIM5α-HA. The amount of plasmid DNA was adjusted to 1.0 µg with pcDNA3.1. Two days post-transfection, viral titer in the culture supernatants was analyzed with TZM-bl indicator cells. (A) Relative viral titer obtained without exogenous SOCS1 and RhTRIM5α was arbitrarily set as 100%. The results are shown as an average of three independent experiments with standard deviation. (B) Whole cell lysates in panel (A) were subjected to immunoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (C) Relative band intensities of RhTRIM5α and SOCS1 in panel (B) were normalized with that of GAPDH. The normalized band intensity of RhTRIM5α (upper panels) in the absence of SOCS1 and that of SOCS1 (lower panels) without exogenous SOCS1 were arbitrarily set as 100%. (D) Effect of SOCS1 expression on transcriptional activity. HEK293T cell were co-transfected with 0.05 µg of pGL4.84-EF1α-hRlucCP (EF1α) or pcDNA3.1-Luc (CMV) together with 0.45 µg of pcDNA3.1 or pHuSOCS1. The amount of plasmid DNA was adjusted to 0.5 µg with pcDNA3.1. Two days after transfection, luciferase activity was determined and normalized with protein concentration (Luc/protein). The results are shown as an average of three independent experiments with standard deviation.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Expressing, Transfection, Plasmid Preparation, Standard Deviation, Activity Assay, Luciferase, Protein Concentration

    Reduction of RhTRIM5α by SOCS1 is proteasome-independent. HEK293T cells were transfected with 0.1 µg of pNL4-3, 0.3 µg of pRhTRIM5α-HA with or without 0.6 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Twenty-four hours after transfection, cells were treated with 30 µM of MG115 (lanes 3 and 4) or 30 µM of MG132 (lanes 5 and 6) for 16 hours. Whole cell lysates were subjected to immunoblot analyses with anti-HA (RhTRIM5α-HA, top panel), anti-SOCS1 (middle panel), anti-IκBα and anti-GAPDH (as loading control, bottom panel) antibodies.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: Reduction of RhTRIM5α by SOCS1 is proteasome-independent. HEK293T cells were transfected with 0.1 µg of pNL4-3, 0.3 µg of pRhTRIM5α-HA with or without 0.6 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Twenty-four hours after transfection, cells were treated with 30 µM of MG115 (lanes 3 and 4) or 30 µM of MG132 (lanes 5 and 6) for 16 hours. Whole cell lysates were subjected to immunoblot analyses with anti-HA (RhTRIM5α-HA, top panel), anti-SOCS1 (middle panel), anti-IκBα and anti-GAPDH (as loading control, bottom panel) antibodies.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Transfection

    SOCS1 reverses RhTRIM5α-mediated late restriction of HIV-1 replication. (A) HEK293T cells were transfected with 0.1 µg of pNL4-3 and with or without 0.6 µg of pHuSOCS1 (S1) and/or 0.3 µg of pRhTRIM5α-HA (T5α). Ct represents transfection with the control vectors. T5α/S1 indicates that S1 and T5α were co-transfected besides pNL4-3. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Two days post-transfection, relative viral titer in the supernatants was analyzed with TZM-bl indicator cells. Average of results from four independent experiments is shown with standard deviation. (B) The amount of p24 antigen in the supernatants was quantified with p24-specific ELISA. Data were obtained from the same experimental sets shown in panel A. (C) Twenty-four μg of whole cell lysates were subjected to immnoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (D) Relative RhTRIM5α protein expression level was determined by densitometry analysis in panel (C). The band intensity for T5α was arbitrarily set as 100%. (E) Relative Rh trim5α mRNA expression determined by quantitative RT-PCR. The value for T5α was arbitrarily set as 100%. The results are shown as an average obtained in four independent experiments with standard deviation.

    Journal: PLoS ONE

    Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

    doi: 10.1371/journal.pone.0109640

    Figure Lengend Snippet: SOCS1 reverses RhTRIM5α-mediated late restriction of HIV-1 replication. (A) HEK293T cells were transfected with 0.1 µg of pNL4-3 and with or without 0.6 µg of pHuSOCS1 (S1) and/or 0.3 µg of pRhTRIM5α-HA (T5α). Ct represents transfection with the control vectors. T5α/S1 indicates that S1 and T5α were co-transfected besides pNL4-3. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Two days post-transfection, relative viral titer in the supernatants was analyzed with TZM-bl indicator cells. Average of results from four independent experiments is shown with standard deviation. (B) The amount of p24 antigen in the supernatants was quantified with p24-specific ELISA. Data were obtained from the same experimental sets shown in panel A. (C) Twenty-four μg of whole cell lysates were subjected to immnoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (D) Relative RhTRIM5α protein expression level was determined by densitometry analysis in panel (C). The band intensity for T5α was arbitrarily set as 100%. (E) Relative Rh trim5α mRNA expression determined by quantitative RT-PCR. The value for T5α was arbitrarily set as 100%. The results are shown as an average obtained in four independent experiments with standard deviation.

    Article Snippet: The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1.

    Techniques: Transfection, Standard Deviation, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-018-03096-0

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

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

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

    UCA1 acted as a ceRNA of miR129 to enhance target SOX4 gene expression in RCC cells. Notes: ( A ) 786O cells were transfected with miR129 mimic or miR-Con, and ACHN cells were transfected with miR129 inhibitor or anti-miR-Con, followed by the detection of SOX4 protein expression. ( B ) 786O cells were transfected with siCon or siUCA1-2, and ACHN cells were transfected with pcDNA3.1 vector or pcDNA-UCA1 plasmid, followed by the determination of SOX4 protein expression. ( C ) Effects of miR129 and UCA1 overexpression on luciferase activity of SOX4 reporter were evaluated in 786O cells. ( D ) The effect of miR129 downregulation and UCA1 silencing on luciferase activity of SOX4 reporter was monitored in ACHN cells. ( E ) SOX4 mRNA expression in 40 pairs of RCC tumor tissue and adjacent normal tissue. ( F ) Spearman’s correlation analysis of SOX4 mRNA and UCA1 expressions in 40 cases of RCC tumor tissue. * P

    Journal: OncoTargets and therapy

    Article Title: UCA1 promotes cell proliferation and invasion and inhibits apoptosis through regulation of the miR129–SOX4 pathway in renal cell carcinoma

    doi: 10.2147/OTT.S160192

    Figure Lengend Snippet: UCA1 acted as a ceRNA of miR129 to enhance target SOX4 gene expression in RCC cells. Notes: ( A ) 786O cells were transfected with miR129 mimic or miR-Con, and ACHN cells were transfected with miR129 inhibitor or anti-miR-Con, followed by the detection of SOX4 protein expression. ( B ) 786O cells were transfected with siCon or siUCA1-2, and ACHN cells were transfected with pcDNA3.1 vector or pcDNA-UCA1 plasmid, followed by the determination of SOX4 protein expression. ( C ) Effects of miR129 and UCA1 overexpression on luciferase activity of SOX4 reporter were evaluated in 786O cells. ( D ) The effect of miR129 downregulation and UCA1 silencing on luciferase activity of SOX4 reporter was monitored in ACHN cells. ( E ) SOX4 mRNA expression in 40 pairs of RCC tumor tissue and adjacent normal tissue. ( F ) Spearman’s correlation analysis of SOX4 mRNA and UCA1 expressions in 40 cases of RCC tumor tissue. * P

    Article Snippet: Full-length sequences of UCA1 were amplified by polymerase chain reaction (PCR) and inserted into pcDNA3.1 vector (Thermo Fisher Scientific) to gain the UCA1-overexpression plasmid.

    Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression, Luciferase, Activity Assay

    UCA1 inhibited miR129 expression in RCC cells by direct interaction. Notes: ( A ) Exhibition of putative binding sites between UCA1 and miR129 and mutant (Mut) sites in UCA1-Mut reporter. ( B ) 786O cells were cotransfected with miR control (Con) or miR129 mimic and wild-type (WT) UCA1 or UCA1-Mut reporter, followed by the detection of luciferase activity at 48 hours posttransfection. ( C ) Luciferase activity was measured in ACHN cells cotransfected with anti-miR-Con or miR129 inhibitor and UCA1-WT or UCA1-Mut reporter at 48 hours after transfection. ( D ) RNA pull-down assays were used to further validate the interaction between UCA1 and miR129 in 786O and ACHN cells. ( E , F ) RNA-immunoprecipitation assays were employed using Ago2 or IgG antibody to explore the enrichment degrees of UCA1 and miR129 in Ago2 or IgG immunoprecipitation complexes of 786O and ACHN cells. The input group acted as a positive control. ( G , H ) miR129 expression was determined in 786O and ACHN cells transfected with siCon ( G ), siUCA1-2 ( G ), UCA1 overexpression plasmid ( H ) or pcDNA3.1 empty vector ( H ). ( I ) miR129 expression analysis in 40 pairs of RCC tumor tissue and adjacent normal tissue. ( J ) Spearman’s correlation analysis of miR129 and UCA1 expression in 40 cases of RCC tumor tissue. * P

    Journal: OncoTargets and therapy

    Article Title: UCA1 promotes cell proliferation and invasion and inhibits apoptosis through regulation of the miR129–SOX4 pathway in renal cell carcinoma

    doi: 10.2147/OTT.S160192

    Figure Lengend Snippet: UCA1 inhibited miR129 expression in RCC cells by direct interaction. Notes: ( A ) Exhibition of putative binding sites between UCA1 and miR129 and mutant (Mut) sites in UCA1-Mut reporter. ( B ) 786O cells were cotransfected with miR control (Con) or miR129 mimic and wild-type (WT) UCA1 or UCA1-Mut reporter, followed by the detection of luciferase activity at 48 hours posttransfection. ( C ) Luciferase activity was measured in ACHN cells cotransfected with anti-miR-Con or miR129 inhibitor and UCA1-WT or UCA1-Mut reporter at 48 hours after transfection. ( D ) RNA pull-down assays were used to further validate the interaction between UCA1 and miR129 in 786O and ACHN cells. ( E , F ) RNA-immunoprecipitation assays were employed using Ago2 or IgG antibody to explore the enrichment degrees of UCA1 and miR129 in Ago2 or IgG immunoprecipitation complexes of 786O and ACHN cells. The input group acted as a positive control. ( G , H ) miR129 expression was determined in 786O and ACHN cells transfected with siCon ( G ), siUCA1-2 ( G ), UCA1 overexpression plasmid ( H ) or pcDNA3.1 empty vector ( H ). ( I ) miR129 expression analysis in 40 pairs of RCC tumor tissue and adjacent normal tissue. ( J ) Spearman’s correlation analysis of miR129 and UCA1 expression in 40 cases of RCC tumor tissue. * P

    Article Snippet: Full-length sequences of UCA1 were amplified by polymerase chain reaction (PCR) and inserted into pcDNA3.1 vector (Thermo Fisher Scientific) to gain the UCA1-overexpression plasmid.

    Techniques: Expressing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection, Immunoprecipitation, Positive Control, Over Expression, Plasmid Preparation