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  • 99
    Thermo Fisher pcdna3 1
    Sox2 suppresses the Wnt/β-catenin signaling activity in A549 and A549/DDP cells. A549 and A549/DDP were transfected with canonical Wnt signaling reporter BATflash and a plasmid expressing Renilla luciferase, along with a plasmid expressing Sox2 or shSox2, or a <t>pcDNA3.1</t> plasmid for 24 h. The cells were then harvested for analysis of luciferase activity and the expression of key components of Wnt/β-catenin signaling cascade. (A) Wnt/β-catenin signaling luciferase reporter demonstrates that Sox2 may inhibit Wnt signaling activity in A549 and A549/DDP cells (P
    Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 52985 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna3 1 expression vector
    Sox2 suppresses the Wnt/β-catenin signaling activity in A549 and A549/DDP cells. A549 and A549/DDP were transfected with canonical Wnt signaling reporter BATflash and a plasmid expressing Renilla luciferase, along with a plasmid expressing Sox2 or shSox2, or a <t>pcDNA3.1</t> plasmid for 24 h. The cells were then harvested for analysis of luciferase activity and the expression of key components of Wnt/β-catenin signaling cascade. (A) Wnt/β-catenin signaling luciferase reporter demonstrates that Sox2 may inhibit Wnt signaling activity in A549 and A549/DDP cells (P
    Pcdna3 1 Expression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3644 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Addgene inc pcdna3 1
    Sox2 suppresses the Wnt/β-catenin signaling activity in A549 and A549/DDP cells. A549 and A549/DDP were transfected with canonical Wnt signaling reporter BATflash and a plasmid expressing Renilla luciferase, along with a plasmid expressing Sox2 or shSox2, or a <t>pcDNA3.1</t> plasmid for 24 h. The cells were then harvested for analysis of luciferase activity and the expression of key components of Wnt/β-catenin signaling cascade. (A) Wnt/β-catenin signaling luciferase reporter demonstrates that Sox2 may inhibit Wnt signaling activity in A549 and A549/DDP cells (P
    Pcdna3 1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 970 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna
    Sox2 suppresses the Wnt/β-catenin signaling activity in A549 and A549/DDP cells. A549 and A549/DDP were transfected with canonical Wnt signaling reporter BATflash and a plasmid expressing Renilla luciferase, along with a plasmid expressing Sox2 or shSox2, or a <t>pcDNA3.1</t> plasmid for 24 h. The cells were then harvested for analysis of luciferase activity and the expression of key components of Wnt/β-catenin signaling cascade. (A) Wnt/β-catenin signaling luciferase reporter demonstrates that Sox2 may inhibit Wnt signaling activity in A549 and A549/DDP cells (P
    Pcdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher eukaryotic expression vector pcdna3 1
    CAPA-1 NMU-like neuropeptides signal from ASG chemosensory neurons to promote gustatory learning through NMUR-1 (a) Luminescence-based calcium mobilization assay for measuring GPCR activation. NMUR-1 is expressed in CHO cells that stably co-express the promiscuous human Gα 16 subunit, which couples receptor activation to calcium release from intracellular storage sites. Intracellular calcium levels are monitored using the calcium indicator aequorin. (b) Calcium responses of CHO cells expressing NMUR-1 are shown relative (%) to the highest value (100% activation) after normalization to the total calcium response. Cells transfected with an empty <t>pcDNA3.1</t> vector are used as a control. Error bars show S.E.M (n ≥ 6). (c) The capa-1/nlp-44 gene encodes a neuropeptide precursor that harbors three predicted peptide sequences (CAPA-1-1, -2, and -3), which are flanked by mono- and dibasic cleaving sites. Black bars indicate the positions of deletions in capa-1/nlp-44 mutant alleles used in this study. (d) Sequence comparison of C. elegans CAPA-1 neuropeptides and NmU neuropeptides from H. sapiens , D. rerio , and D. melanogaster . Conserved C-terminal features are highlighted in black. Residues with similar physicochemical properties are colored gray. (e) Mean position on the gradient through time and (g) chemotactic indices of two independent deletion mutants of the capa-1/nlp-44 neuropeptide precursor gene after mock- and NaCl-conditioning. The corresponding biased random walk and klinotaxis indices are shown on Supplementary Fig. 5a . n ≥ 25 animals per genotype. (f) Co-localization of a bicistronic GFP construct harboring the promoter, genomic DNA and 3’UTR of the capa-1 gene (capa-1p::capa-1::SL2::gfp ) with mCherry expression from the ASG-specific gcy-15 promotor 55 . The asterisk marks fluorescence in the intestine from the elt-2p::gfp co-injection marker. Scale bar, 35 µm. (h) Chemotactic behavior of NaCl-conditioned capa-1 (ok3065) animals in which wild type copies of the capa-1 gene are reintroduced under the control of its promoter sequence ( capa-1p::capa-1::SL2::gfp ). The corresponding mean population position on the gradient, biased random walk and klinotaxis indices are shown on Supplementary Fig. 5b . n ≥ 37 animals per genotype. (i) Chemotactic behavior of NaCl-conditioned animals after ASG-specific RNAi-mediated knockdown of capa-1 , achieved by expressing sense and anti-sense capa-1 sequences under the control of the ASG-specific ops-1 promoter 60 . A strain carrying only the elt-2p ::GFP co-injection marker is used as a control to exclude potential effects of the transgene selection marker. The corresponding mean population position on the gradient, biased random walk and klinotaxis indices are shown on Supplementary Fig. 5c . n ≥ 27 animals per genotype.
    Eukaryotic Expression Vector Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene pcdna3 1
    A46R associates with MyD88 and blocks MyD88-dependent signaling. (a) Alignment of A46R with human TIR domains. The conserved motifs Box 1, Box 2, and Box 3 are indicated by a solid line. The eight differences between VV and variola virus A46R sequences are indicated by an asterisk. For A46R, amino acids 35–238 are shown. (b) HEK 293T cells were transfected with A46R and AU1-MyD88 (left) or Flag-TRAF2 (right) as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (c) HEK 293T cells were transfected with 8 μg AU1-MyD88 (top) or Flag-TRAF2 (bottom). After 24 h, lysates were incubated with GST alone (lane 2) or GST-A46R (lane 3), and together with whole cell lysates (lane 1) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (d) HEK 293 cells were transfected with 4 μg of myc-MyD88. After 24 h, cells were infected with viruses either containing (vWT-A46R) or not (vΔA46R) the A46R gene (MOI = 1) and harvested 24 h after infection. Lysates were subjected to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. Whole cell lysates were analyzed for expression of A46R. (e) Murine macrophage RAW 264.7 cells were transfected with the phRL-TK reporter gene and the NF-κB luciferase construct as described in Materials and methods, together with <t>pcDNA3.1</t> or 100 ng A46R. Cells were stimulated for 6 h with 10 nM MALP-2 (MALP), 5 μg/ml Pam3Cys (Pam), 1 μg/ml LPS, 250 ng/ml Flagellin (Flag), 1 μM R-848, or 5 μg/ml CpG DNA. Cells were harvested 24 h after transfection and the reporter gene activity was measured.
    Pcdna3 1, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 885 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pcdna3 1 hygro
    A46R associates with MyD88 and blocks MyD88-dependent signaling. (a) Alignment of A46R with human TIR domains. The conserved motifs Box 1, Box 2, and Box 3 are indicated by a solid line. The eight differences between VV and variola virus A46R sequences are indicated by an asterisk. For A46R, amino acids 35–238 are shown. (b) HEK 293T cells were transfected with A46R and AU1-MyD88 (left) or Flag-TRAF2 (right) as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (c) HEK 293T cells were transfected with 8 μg AU1-MyD88 (top) or Flag-TRAF2 (bottom). After 24 h, lysates were incubated with GST alone (lane 2) or GST-A46R (lane 3), and together with whole cell lysates (lane 1) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (d) HEK 293 cells were transfected with 4 μg of myc-MyD88. After 24 h, cells were infected with viruses either containing (vWT-A46R) or not (vΔA46R) the A46R gene (MOI = 1) and harvested 24 h after infection. Lysates were subjected to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. Whole cell lysates were analyzed for expression of A46R. (e) Murine macrophage RAW 264.7 cells were transfected with the phRL-TK reporter gene and the NF-κB luciferase construct as described in Materials and methods, together with <t>pcDNA3.1</t> or 100 ng A46R. Cells were stimulated for 6 h with 10 nM MALP-2 (MALP), 5 μg/ml Pam3Cys (Pam), 1 μg/ml LPS, 250 ng/ml Flagellin (Flag), 1 μM R-848, or 5 μg/ml CpG DNA. Cells were harvested 24 h after transfection and the reporter gene activity was measured.
    Pcdna3 1 Hygro, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 902 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher pcdna3 1 zeo
    A46R associates with MyD88 and blocks MyD88-dependent signaling. (a) Alignment of A46R with human TIR domains. The conserved motifs Box 1, Box 2, and Box 3 are indicated by a solid line. The eight differences between VV and variola virus A46R sequences are indicated by an asterisk. For A46R, amino acids 35–238 are shown. (b) HEK 293T cells were transfected with A46R and AU1-MyD88 (left) or Flag-TRAF2 (right) as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (c) HEK 293T cells were transfected with 8 μg AU1-MyD88 (top) or Flag-TRAF2 (bottom). After 24 h, lysates were incubated with GST alone (lane 2) or GST-A46R (lane 3), and together with whole cell lysates (lane 1) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (d) HEK 293 cells were transfected with 4 μg of myc-MyD88. After 24 h, cells were infected with viruses either containing (vWT-A46R) or not (vΔA46R) the A46R gene (MOI = 1) and harvested 24 h after infection. Lysates were subjected to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. Whole cell lysates were analyzed for expression of A46R. (e) Murine macrophage RAW 264.7 cells were transfected with the phRL-TK reporter gene and the NF-κB luciferase construct as described in Materials and methods, together with <t>pcDNA3.1</t> or 100 ng A46R. Cells were stimulated for 6 h with 10 nM MALP-2 (MALP), 5 μg/ml Pam3Cys (Pam), 1 μg/ml LPS, 250 ng/ml Flagellin (Flag), 1 μM R-848, or 5 μg/ml CpG DNA. Cells were harvested 24 h after transfection and the reporter gene activity was measured.
    Pcdna3 1 Zeo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 859 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GenScript pcdna3 1
    A46R associates with MyD88 and blocks MyD88-dependent signaling. (a) Alignment of A46R with human TIR domains. The conserved motifs Box 1, Box 2, and Box 3 are indicated by a solid line. The eight differences between VV and variola virus A46R sequences are indicated by an asterisk. For A46R, amino acids 35–238 are shown. (b) HEK 293T cells were transfected with A46R and AU1-MyD88 (left) or Flag-TRAF2 (right) as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (c) HEK 293T cells were transfected with 8 μg AU1-MyD88 (top) or Flag-TRAF2 (bottom). After 24 h, lysates were incubated with GST alone (lane 2) or GST-A46R (lane 3), and together with whole cell lysates (lane 1) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (d) HEK 293 cells were transfected with 4 μg of myc-MyD88. After 24 h, cells were infected with viruses either containing (vWT-A46R) or not (vΔA46R) the A46R gene (MOI = 1) and harvested 24 h after infection. Lysates were subjected to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. Whole cell lysates were analyzed for expression of A46R. (e) Murine macrophage RAW 264.7 cells were transfected with the phRL-TK reporter gene and the NF-κB luciferase construct as described in Materials and methods, together with <t>pcDNA3.1</t> or 100 ng A46R. Cells were stimulated for 6 h with 10 nM MALP-2 (MALP), 5 μg/ml Pam3Cys (Pam), 1 μg/ml LPS, 250 ng/ml Flagellin (Flag), 1 μM R-848, or 5 μg/ml CpG DNA. Cells were harvested 24 h after transfection and the reporter gene activity was measured.
    Pcdna3 1, supplied by GenScript, used in various techniques. Bioz Stars score: 93/100, based on 627 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher pcdna3 1 myc
    NP expression in mammalian cells leads to downreglation of PKR and eIF2α phosphorylation. HEK293T cells were transfected with NP expressing plasmid <t>(pcDNA3.1-NP)</t> or control plasmid (pcDNA3.1). Cells were harvested at 36 hours post-transfection and cell lysates were subjected to western blotting analysis. A. Lane 1 of panel 2 shows significant downregulation of pPKR levels in NP transfected cells. Panel 1 shows NP expression level, whereas panel 3 shows equal loading trough β-Actin control. B. Figure shows graphical representation of relative pPKR levels as measured by western blotting followed by densitometric measurement in 3 independent experiments. Error bars represent standard deviation. C. Lane 1 of panel 2 shows significant downregulation of p-eIF2α levels in NP transfected cells. Panel 1 shows NP expression level, whereas panel 3 shows equal loading trough β-Actin control. D. Figure shows graphical representation of relative p-eIF2α levels as measured by western blotting followed by densitometric measurement in 3 independent experiments. Error bars represent standard deviation.
    Pcdna3 1 Myc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 737 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sox2 suppresses the Wnt/β-catenin signaling activity in A549 and A549/DDP cells. A549 and A549/DDP were transfected with canonical Wnt signaling reporter BATflash and a plasmid expressing Renilla luciferase, along with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 24 h. The cells were then harvested for analysis of luciferase activity and the expression of key components of Wnt/β-catenin signaling cascade. (A) Wnt/β-catenin signaling luciferase reporter demonstrates that Sox2 may inhibit Wnt signaling activity in A549 and A549/DDP cells (P

    Journal: Molecular Medicine Reports

    Article Title: Sox2 inhibits Wnt-β-catenin signaling and metastatic potency of cisplatin-resistant lung adenocarcinoma cells

    doi: 10.3892/mmr.2017.6170

    Figure Lengend Snippet: Sox2 suppresses the Wnt/β-catenin signaling activity in A549 and A549/DDP cells. A549 and A549/DDP were transfected with canonical Wnt signaling reporter BATflash and a plasmid expressing Renilla luciferase, along with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 24 h. The cells were then harvested for analysis of luciferase activity and the expression of key components of Wnt/β-catenin signaling cascade. (A) Wnt/β-catenin signaling luciferase reporter demonstrates that Sox2 may inhibit Wnt signaling activity in A549 and A549/DDP cells (P

    Article Snippet: The pcDNA3.1 plasmid (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was always included as a control.

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

    Sox2 enhances the stemness of lung cancer cells determined by a clonogenic assay. A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid and their capacity for clone formation was analyzed using a clonogenic assay in 35 mm dishes. (A) Representative images of clonogenic assay for A549 cells (left) and its relevant quantification of the number of colonies (right). (B) Representative images of clonogenic assay for A549/DDP cells (left) and its relevant quantification of the number of colonies (right). An overexpression of Sox2 demonstrated an ability to enhance the clone formation in A549 and A549/DDP cells, and a shRNA-mediated knockdown of Sox2 marginally reduced the clone formation. Data represented the mean ± standard deviation from three independent triplicated experiments (n=9). **P

    Journal: Molecular Medicine Reports

    Article Title: Sox2 inhibits Wnt-β-catenin signaling and metastatic potency of cisplatin-resistant lung adenocarcinoma cells

    doi: 10.3892/mmr.2017.6170

    Figure Lengend Snippet: Sox2 enhances the stemness of lung cancer cells determined by a clonogenic assay. A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid and their capacity for clone formation was analyzed using a clonogenic assay in 35 mm dishes. (A) Representative images of clonogenic assay for A549 cells (left) and its relevant quantification of the number of colonies (right). (B) Representative images of clonogenic assay for A549/DDP cells (left) and its relevant quantification of the number of colonies (right). An overexpression of Sox2 demonstrated an ability to enhance the clone formation in A549 and A549/DDP cells, and a shRNA-mediated knockdown of Sox2 marginally reduced the clone formation. Data represented the mean ± standard deviation from three independent triplicated experiments (n=9). **P

    Article Snippet: The pcDNA3.1 plasmid (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was always included as a control.

    Techniques: Clonogenic Assay, Transfection, Plasmid Preparation, Expressing, Over Expression, shRNA, Standard Deviation

    Sox2 inhibits cisplatin-induced apoptosis in lung cancer cells. A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 12 h, and then cultured in medium containing 10 µM cisplatin for an additional 24 h prior to being harvested for analysis. (A) MTT assay determined the proliferation of cells in the presence of cisplatin. The transient transduction of Sox2 or shSox2 had no effect on cell proliferation. Overexpression of Sox2 increased the survival rate of A549 cells in the presence of cisplatin, but had no effect on cisplatin-resistant A59/DDP cells. Notably, inhibition of Sox2 expression by short hairpin RNA increased the cisplatin-induced cell death in A549/DDP cells. (B) Cell apoptosis analyzed by a cytometric assay. An inhibition of Sox2 by shSox2 significantly enhanced cisplatin-induced apoptosis in A549 and A549/DDP cells (P

    Journal: Molecular Medicine Reports

    Article Title: Sox2 inhibits Wnt-β-catenin signaling and metastatic potency of cisplatin-resistant lung adenocarcinoma cells

    doi: 10.3892/mmr.2017.6170

    Figure Lengend Snippet: Sox2 inhibits cisplatin-induced apoptosis in lung cancer cells. A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 12 h, and then cultured in medium containing 10 µM cisplatin for an additional 24 h prior to being harvested for analysis. (A) MTT assay determined the proliferation of cells in the presence of cisplatin. The transient transduction of Sox2 or shSox2 had no effect on cell proliferation. Overexpression of Sox2 increased the survival rate of A549 cells in the presence of cisplatin, but had no effect on cisplatin-resistant A59/DDP cells. Notably, inhibition of Sox2 expression by short hairpin RNA increased the cisplatin-induced cell death in A549/DDP cells. (B) Cell apoptosis analyzed by a cytometric assay. An inhibition of Sox2 by shSox2 significantly enhanced cisplatin-induced apoptosis in A549 and A549/DDP cells (P

    Article Snippet: The pcDNA3.1 plasmid (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was always included as a control.

    Techniques: Transfection, Plasmid Preparation, Expressing, Cell Culture, MTT Assay, Transduction, Over Expression, Inhibition, shRNA

    Apoptosis associated proteins determined by an immunoblotting analysis. A549 and A549/DDP cells were transfected with plasmid expressing Sox2 or shSox2, or control pcDNA3.1 plasmid for 12 h, and then cultured in medium containing 10 µM cisplatin for additional 24 h prior to being harvested for immunoblotting analysis for indicated proteins. The values labeled on the top of each bands represented the relative expression levels of proteins over their respective pcDNA3.1 control as determined by a densitometric assay. Overexpression of Sox2 demonstrated a trend to reduce the expression of pro-apoptotic proteins (caspase-3, Bax), but increased the expression of anti-apoptotic proteins Bcl-2 in lung cancer cells. Cas 3: caspase-3; Bax, Bcl-2-like protein 4; AIF, apoptosis inducing factor; Bcl-2, B-cell lymphoma 2; Mcl-1, myeloid cell leukemia sequence 1 protein; C, control; Sox2, sex-determining region Y box 2; shSox2, Sox2 short hairpin RNA.

    Journal: Molecular Medicine Reports

    Article Title: Sox2 inhibits Wnt-β-catenin signaling and metastatic potency of cisplatin-resistant lung adenocarcinoma cells

    doi: 10.3892/mmr.2017.6170

    Figure Lengend Snippet: Apoptosis associated proteins determined by an immunoblotting analysis. A549 and A549/DDP cells were transfected with plasmid expressing Sox2 or shSox2, or control pcDNA3.1 plasmid for 12 h, and then cultured in medium containing 10 µM cisplatin for additional 24 h prior to being harvested for immunoblotting analysis for indicated proteins. The values labeled on the top of each bands represented the relative expression levels of proteins over their respective pcDNA3.1 control as determined by a densitometric assay. Overexpression of Sox2 demonstrated a trend to reduce the expression of pro-apoptotic proteins (caspase-3, Bax), but increased the expression of anti-apoptotic proteins Bcl-2 in lung cancer cells. Cas 3: caspase-3; Bax, Bcl-2-like protein 4; AIF, apoptosis inducing factor; Bcl-2, B-cell lymphoma 2; Mcl-1, myeloid cell leukemia sequence 1 protein; C, control; Sox2, sex-determining region Y box 2; shSox2, Sox2 short hairpin RNA.

    Article Snippet: The pcDNA3.1 plasmid (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was always included as a control.

    Techniques: Transfection, Plasmid Preparation, Expressing, Cell Culture, Labeling, Over Expression, Sequencing, shRNA

    Effect of Sox2 on the invasion of lung cancer cells in vitro . A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 12 h, the capability of cell invasion was accessed by a Transwell analysis, and cell proliferative ability was ascertained by an MTT assay. (A) Representative images of Transwell assay for A549 cells (left) and its relevant quantification of the numbers of invasive cells (right). (B) Representative images of Transwell assay for A549/DDP cells (left) and its relevant quantification of the numbers of invasive cells (right). Overexpression of Sox2 enhanced cell invasion in A549 cells, but had no effect on A549/DDP cells. By contrast, an inhibition of Sox2 by shSox2 promoted cell invasion in A549/DDP cells. Data are presented as the mean ± standard deviation from three independent triplicated experiments (n=9). **P

    Journal: Molecular Medicine Reports

    Article Title: Sox2 inhibits Wnt-β-catenin signaling and metastatic potency of cisplatin-resistant lung adenocarcinoma cells

    doi: 10.3892/mmr.2017.6170

    Figure Lengend Snippet: Effect of Sox2 on the invasion of lung cancer cells in vitro . A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 12 h, the capability of cell invasion was accessed by a Transwell analysis, and cell proliferative ability was ascertained by an MTT assay. (A) Representative images of Transwell assay for A549 cells (left) and its relevant quantification of the numbers of invasive cells (right). (B) Representative images of Transwell assay for A549/DDP cells (left) and its relevant quantification of the numbers of invasive cells (right). Overexpression of Sox2 enhanced cell invasion in A549 cells, but had no effect on A549/DDP cells. By contrast, an inhibition of Sox2 by shSox2 promoted cell invasion in A549/DDP cells. Data are presented as the mean ± standard deviation from three independent triplicated experiments (n=9). **P

    Article Snippet: The pcDNA3.1 plasmid (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was always included as a control.

    Techniques: In Vitro, Transfection, Plasmid Preparation, Expressing, MTT Assay, Transwell Assay, Over Expression, Inhibition, Standard Deviation

    Effect of Sox2 on the migration of lung cancer cells in vitro . A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 12 h and the capability of cell migration was assessed using a scratch assay. (A) Representative images of scratch assays for A549 cells (left panel) and its relevant quantification of the results of cell migration index (right panel). (B) Representative images of scratch assays for A549/DDP cells (left panel) and its relevant quantification of the results of cell migration index (right panel). Overexpression of Sox2 enhanced cell migration in A549 cells, but had no effect on A549/DDP cells. By contrast, inhibition of Sox2 by shSox2 promoted cell migration in A549/DDP cells. *P

    Journal: Molecular Medicine Reports

    Article Title: Sox2 inhibits Wnt-β-catenin signaling and metastatic potency of cisplatin-resistant lung adenocarcinoma cells

    doi: 10.3892/mmr.2017.6170

    Figure Lengend Snippet: Effect of Sox2 on the migration of lung cancer cells in vitro . A549 and A549/DDP cells were transfected with a plasmid expressing Sox2 or shSox2, or a pcDNA3.1 plasmid for 12 h and the capability of cell migration was assessed using a scratch assay. (A) Representative images of scratch assays for A549 cells (left panel) and its relevant quantification of the results of cell migration index (right panel). (B) Representative images of scratch assays for A549/DDP cells (left panel) and its relevant quantification of the results of cell migration index (right panel). Overexpression of Sox2 enhanced cell migration in A549 cells, but had no effect on A549/DDP cells. By contrast, inhibition of Sox2 by shSox2 promoted cell migration in A549/DDP cells. *P

    Article Snippet: The pcDNA3.1 plasmid (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was always included as a control.

    Techniques: Migration, In Vitro, Transfection, Plasmid Preparation, Expressing, Wound Healing Assay, Over Expression, Inhibition

    Evaluation of sialidase NEU3 effect on cell viability with or without gefitinib. MTT test was performed on HSAEC1, HCC4006 and H1734 cell lines transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3 and then treated or not with 27 nM or 1 μM gefitinib for 36 h post -transfection. Data were normalized on control cells transfected with the empty vector (A). Cell viabilities of mock and NEU3 transfected cells are reported after treatment with either 27 nM (B) or 1 μM gefitinib (C). Data were normalized on control cells without drug. Values are presented as means ± standard error (SE).

    Journal: PLoS ONE

    Article Title: Non-small cell lung cancer (NSCLC), EGFR downstream pathway activation and TKI targeted therapies sensitivity: Effect of the plasma membrane-associated NEU3

    doi: 10.1371/journal.pone.0187289

    Figure Lengend Snippet: Evaluation of sialidase NEU3 effect on cell viability with or without gefitinib. MTT test was performed on HSAEC1, HCC4006 and H1734 cell lines transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3 and then treated or not with 27 nM or 1 μM gefitinib for 36 h post -transfection. Data were normalized on control cells transfected with the empty vector (A). Cell viabilities of mock and NEU3 transfected cells are reported after treatment with either 27 nM (B) or 1 μM gefitinib (C). Data were normalized on control cells without drug. Values are presented as means ± standard error (SE).

    Article Snippet: Vector cDNA coding for human sialidase NEU3 was previously subcloned into plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), in frame with C-terminal hemagglutinin (HA) epitope [ ].

    Techniques: MTT Assay, Transfection, Plasmid Preparation

    NEU3 transfection evaluation and NEU3 membrane localization. (A) Relative quantification of NEU3 mRNA levels by qPCR on HSAEC1, HCC4006 and H1734 cell lines transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3. (B) Representative Western-blot analyses performed on membrane fractions. Proteins were separated on a 10% SDS-PAGE and probed with anti-NEU3 antibody. Transferrin receptor was used as a membrane marker. The experiments were performed in quadruplicate. (C) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after transfection with pcDNA3.1-HsNEU3 with those obtained after transfection with the empty vector (mock). Values are presented as means ± standard error (SE). ***p

    Journal: PLoS ONE

    Article Title: Non-small cell lung cancer (NSCLC), EGFR downstream pathway activation and TKI targeted therapies sensitivity: Effect of the plasma membrane-associated NEU3

    doi: 10.1371/journal.pone.0187289

    Figure Lengend Snippet: NEU3 transfection evaluation and NEU3 membrane localization. (A) Relative quantification of NEU3 mRNA levels by qPCR on HSAEC1, HCC4006 and H1734 cell lines transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3. (B) Representative Western-blot analyses performed on membrane fractions. Proteins were separated on a 10% SDS-PAGE and probed with anti-NEU3 antibody. Transferrin receptor was used as a membrane marker. The experiments were performed in quadruplicate. (C) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after transfection with pcDNA3.1-HsNEU3 with those obtained after transfection with the empty vector (mock). Values are presented as means ± standard error (SE). ***p

    Article Snippet: Vector cDNA coding for human sialidase NEU3 was previously subcloned into plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), in frame with C-terminal hemagglutinin (HA) epitope [ ].

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation, Western Blot, SDS Page, Marker, Software

    EGFR phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment. (A) Representative Western-blot analyses performed on a normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected either with the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-EGFR, anti-P-EGFR antibodies. GAPDH was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained in the absence of EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p

    Journal: PLoS ONE

    Article Title: Non-small cell lung cancer (NSCLC), EGFR downstream pathway activation and TKI targeted therapies sensitivity: Effect of the plasma membrane-associated NEU3

    doi: 10.1371/journal.pone.0187289

    Figure Lengend Snippet: EGFR phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment. (A) Representative Western-blot analyses performed on a normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected either with the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-EGFR, anti-P-EGFR antibodies. GAPDH was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained in the absence of EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p

    Article Snippet: Vector cDNA coding for human sialidase NEU3 was previously subcloned into plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), in frame with C-terminal hemagglutinin (HA) epitope [ ].

    Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, SDS Page, Software

    Akt phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment. (A) Representative Western-blot analyses performed on normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-Akt, anti-P-Akt antibodies. Vinculin was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained without EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p

    Journal: PLoS ONE

    Article Title: Non-small cell lung cancer (NSCLC), EGFR downstream pathway activation and TKI targeted therapies sensitivity: Effect of the plasma membrane-associated NEU3

    doi: 10.1371/journal.pone.0187289

    Figure Lengend Snippet: Akt phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment. (A) Representative Western-blot analyses performed on normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-Akt, anti-P-Akt antibodies. Vinculin was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained without EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p

    Article Snippet: Vector cDNA coding for human sialidase NEU3 was previously subcloned into plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), in frame with C-terminal hemagglutinin (HA) epitope [ ].

    Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, SDS Page, Software

    ERK phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment. (A) Representative Western-blot analyses performed on normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-ERK1/2, anti-P-ERK1/2 antibodies. Vinculin was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained without EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p

    Journal: PLoS ONE

    Article Title: Non-small cell lung cancer (NSCLC), EGFR downstream pathway activation and TKI targeted therapies sensitivity: Effect of the plasma membrane-associated NEU3

    doi: 10.1371/journal.pone.0187289

    Figure Lengend Snippet: ERK phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment. (A) Representative Western-blot analyses performed on normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-ERK1/2, anti-P-ERK1/2 antibodies. Vinculin was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained without EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p

    Article Snippet: Vector cDNA coding for human sialidase NEU3 was previously subcloned into plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), in frame with C-terminal hemagglutinin (HA) epitope [ ].

    Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, SDS Page, Software

    H. pylori -induced HB-EGF shedding is important for gastrin expression. (A,D) Schematic diagrams of double epitope-tagged membrane-anchored proHBEGF-HA/Myc and Uncleavable proHBEGF-HA/Myc constructs, pHB-EGF, and pUcHB-EGF, respectively. (B) AGS cells were transfected with pcDNA3 (lane 1) or with pHB-EGF (lanes 2–6). Cells expressing proHBEGF-HA/Myc were treated with 1 μM TPA for 30 min, or infected with G27 WT, G27Δ cagA , or G27ΔPAI for 4 h. Existence of shed HB-EGF HA in concentrated conditioned medium (upper panel) and CTF Myc in cell lysate (lower panel) were examined by Western blot with anti-HA and anti-Myc antibody, respectively. Intensities of each HB-EGF HA or CTF Myc band was measured and normalized to intensity of GAPDH. Intensities of each band were then compared to that of the control, and presented as relative band intensity. (C) AGS cells transfected with pHB-EGF were infected with G27 WT or G27ΔPAI for 4 h and proHBEGF-HA/Myc was observed using confocal microscopy. proHBEGF-HA/Myc and actin were stained with green and red fluorescence, respectively. Each image is representative of three independent experiments. The proportion of HB-EGF HA positive cells was obtained by calculating ratio between the green-fluorescent area and the total fluorescent area. The average proportion of HB-EGF HA positive cells was derived from two independent experiments. (E) AGS cells were transfected with pHB-EGF (lane 1) or with pUcHB-EGF (lane 2), and treated with 1 μM TPA for 30 min. Concentrated conditioned medium (upper panel) and cell lysate (lower panel) were analyzed by Western blot to detect HB-EGF HA and CTF Myc , respectively. (F) G240-Luc cells were transfected with pcDNA3, pUcHB-EGF, or pHB-EGF, and subsequently infected with G27 for 4 h. Gastrin promoter activity was measured by luciferase assay. * , #, †, ‡, and § indicates statistically significant difference ( P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Helicobacter pylori-Induced HB-EGF Upregulates Gastrin Expression via the EGF Receptor, C-Raf, Mek1, and Erk2 in the MAPK Pathway

    doi: 10.3389/fcimb.2017.00541

    Figure Lengend Snippet: H. pylori -induced HB-EGF shedding is important for gastrin expression. (A,D) Schematic diagrams of double epitope-tagged membrane-anchored proHBEGF-HA/Myc and Uncleavable proHBEGF-HA/Myc constructs, pHB-EGF, and pUcHB-EGF, respectively. (B) AGS cells were transfected with pcDNA3 (lane 1) or with pHB-EGF (lanes 2–6). Cells expressing proHBEGF-HA/Myc were treated with 1 μM TPA for 30 min, or infected with G27 WT, G27Δ cagA , or G27ΔPAI for 4 h. Existence of shed HB-EGF HA in concentrated conditioned medium (upper panel) and CTF Myc in cell lysate (lower panel) were examined by Western blot with anti-HA and anti-Myc antibody, respectively. Intensities of each HB-EGF HA or CTF Myc band was measured and normalized to intensity of GAPDH. Intensities of each band were then compared to that of the control, and presented as relative band intensity. (C) AGS cells transfected with pHB-EGF were infected with G27 WT or G27ΔPAI for 4 h and proHBEGF-HA/Myc was observed using confocal microscopy. proHBEGF-HA/Myc and actin were stained with green and red fluorescence, respectively. Each image is representative of three independent experiments. The proportion of HB-EGF HA positive cells was obtained by calculating ratio between the green-fluorescent area and the total fluorescent area. The average proportion of HB-EGF HA positive cells was derived from two independent experiments. (E) AGS cells were transfected with pHB-EGF (lane 1) or with pUcHB-EGF (lane 2), and treated with 1 μM TPA for 30 min. Concentrated conditioned medium (upper panel) and cell lysate (lower panel) were analyzed by Western blot to detect HB-EGF HA and CTF Myc , respectively. (F) G240-Luc cells were transfected with pcDNA3, pUcHB-EGF, or pHB-EGF, and subsequently infected with G27 for 4 h. Gastrin promoter activity was measured by luciferase assay. * , #, †, ‡, and § indicates statistically significant difference ( P

    Article Snippet: AGS cells were cotransfected with pG240-Luc and pcDNA3 mammalian expression vector (Invitrogen), and transfectants were selected by 400 μg/ml of G418 (Sigma-Aldrich).

    Techniques: Expressing, Construct, Transfection, Infection, Western Blot, Confocal Microscopy, Staining, Fluorescence, Derivative Assay, Activity Assay, Luciferase

    CAPA-1 NMU-like neuropeptides signal from ASG chemosensory neurons to promote gustatory learning through NMUR-1 (a) Luminescence-based calcium mobilization assay for measuring GPCR activation. NMUR-1 is expressed in CHO cells that stably co-express the promiscuous human Gα 16 subunit, which couples receptor activation to calcium release from intracellular storage sites. Intracellular calcium levels are monitored using the calcium indicator aequorin. (b) Calcium responses of CHO cells expressing NMUR-1 are shown relative (%) to the highest value (100% activation) after normalization to the total calcium response. Cells transfected with an empty pcDNA3.1 vector are used as a control. Error bars show S.E.M (n ≥ 6). (c) The capa-1/nlp-44 gene encodes a neuropeptide precursor that harbors three predicted peptide sequences (CAPA-1-1, -2, and -3), which are flanked by mono- and dibasic cleaving sites. Black bars indicate the positions of deletions in capa-1/nlp-44 mutant alleles used in this study. (d) Sequence comparison of C. elegans CAPA-1 neuropeptides and NmU neuropeptides from H. sapiens , D. rerio , and D. melanogaster . Conserved C-terminal features are highlighted in black. Residues with similar physicochemical properties are colored gray. (e) Mean position on the gradient through time and (g) chemotactic indices of two independent deletion mutants of the capa-1/nlp-44 neuropeptide precursor gene after mock- and NaCl-conditioning. The corresponding biased random walk and klinotaxis indices are shown on Supplementary Fig. 5a . n ≥ 25 animals per genotype. (f) Co-localization of a bicistronic GFP construct harboring the promoter, genomic DNA and 3’UTR of the capa-1 gene (capa-1p::capa-1::SL2::gfp ) with mCherry expression from the ASG-specific gcy-15 promotor 55 . The asterisk marks fluorescence in the intestine from the elt-2p::gfp co-injection marker. Scale bar, 35 µm. (h) Chemotactic behavior of NaCl-conditioned capa-1 (ok3065) animals in which wild type copies of the capa-1 gene are reintroduced under the control of its promoter sequence ( capa-1p::capa-1::SL2::gfp ). The corresponding mean population position on the gradient, biased random walk and klinotaxis indices are shown on Supplementary Fig. 5b . n ≥ 37 animals per genotype. (i) Chemotactic behavior of NaCl-conditioned animals after ASG-specific RNAi-mediated knockdown of capa-1 , achieved by expressing sense and anti-sense capa-1 sequences under the control of the ASG-specific ops-1 promoter 60 . A strain carrying only the elt-2p ::GFP co-injection marker is used as a control to exclude potential effects of the transgene selection marker. The corresponding mean population position on the gradient, biased random walk and klinotaxis indices are shown on Supplementary Fig. 5c . n ≥ 27 animals per genotype.

    Journal: bioRxiv

    Article Title: Neuromedin U signaling regulates memory retrieval of learned salt avoidance in a C. elegans gustatory circuit

    doi: 10.1101/683888

    Figure Lengend Snippet: CAPA-1 NMU-like neuropeptides signal from ASG chemosensory neurons to promote gustatory learning through NMUR-1 (a) Luminescence-based calcium mobilization assay for measuring GPCR activation. NMUR-1 is expressed in CHO cells that stably co-express the promiscuous human Gα 16 subunit, which couples receptor activation to calcium release from intracellular storage sites. Intracellular calcium levels are monitored using the calcium indicator aequorin. (b) Calcium responses of CHO cells expressing NMUR-1 are shown relative (%) to the highest value (100% activation) after normalization to the total calcium response. Cells transfected with an empty pcDNA3.1 vector are used as a control. Error bars show S.E.M (n ≥ 6). (c) The capa-1/nlp-44 gene encodes a neuropeptide precursor that harbors three predicted peptide sequences (CAPA-1-1, -2, and -3), which are flanked by mono- and dibasic cleaving sites. Black bars indicate the positions of deletions in capa-1/nlp-44 mutant alleles used in this study. (d) Sequence comparison of C. elegans CAPA-1 neuropeptides and NmU neuropeptides from H. sapiens , D. rerio , and D. melanogaster . Conserved C-terminal features are highlighted in black. Residues with similar physicochemical properties are colored gray. (e) Mean position on the gradient through time and (g) chemotactic indices of two independent deletion mutants of the capa-1/nlp-44 neuropeptide precursor gene after mock- and NaCl-conditioning. The corresponding biased random walk and klinotaxis indices are shown on Supplementary Fig. 5a . n ≥ 25 animals per genotype. (f) Co-localization of a bicistronic GFP construct harboring the promoter, genomic DNA and 3’UTR of the capa-1 gene (capa-1p::capa-1::SL2::gfp ) with mCherry expression from the ASG-specific gcy-15 promotor 55 . The asterisk marks fluorescence in the intestine from the elt-2p::gfp co-injection marker. Scale bar, 35 µm. (h) Chemotactic behavior of NaCl-conditioned capa-1 (ok3065) animals in which wild type copies of the capa-1 gene are reintroduced under the control of its promoter sequence ( capa-1p::capa-1::SL2::gfp ). The corresponding mean population position on the gradient, biased random walk and klinotaxis indices are shown on Supplementary Fig. 5b . n ≥ 37 animals per genotype. (i) Chemotactic behavior of NaCl-conditioned animals after ASG-specific RNAi-mediated knockdown of capa-1 , achieved by expressing sense and anti-sense capa-1 sequences under the control of the ASG-specific ops-1 promoter 60 . A strain carrying only the elt-2p ::GFP co-injection marker is used as a control to exclude potential effects of the transgene selection marker. The corresponding mean population position on the gradient, biased random walk and klinotaxis indices are shown on Supplementary Fig. 5c . n ≥ 27 animals per genotype.

    Article Snippet: For heterologous expression of NMUR-1 in Chinese hamster ovary (CHO) cells, nmur-1 cDNA was amplified by PCR from cDNA of mixed-stage wild-type C. elegans and directionally cloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen).

    Techniques: Calcium Mobilization Assay, Activation Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Sequencing, Construct, Fluorescence, Injection, Marker, Selection

    A46R associates with MyD88 and blocks MyD88-dependent signaling. (a) Alignment of A46R with human TIR domains. The conserved motifs Box 1, Box 2, and Box 3 are indicated by a solid line. The eight differences between VV and variola virus A46R sequences are indicated by an asterisk. For A46R, amino acids 35–238 are shown. (b) HEK 293T cells were transfected with A46R and AU1-MyD88 (left) or Flag-TRAF2 (right) as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (c) HEK 293T cells were transfected with 8 μg AU1-MyD88 (top) or Flag-TRAF2 (bottom). After 24 h, lysates were incubated with GST alone (lane 2) or GST-A46R (lane 3), and together with whole cell lysates (lane 1) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (d) HEK 293 cells were transfected with 4 μg of myc-MyD88. After 24 h, cells were infected with viruses either containing (vWT-A46R) or not (vΔA46R) the A46R gene (MOI = 1) and harvested 24 h after infection. Lysates were subjected to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. Whole cell lysates were analyzed for expression of A46R. (e) Murine macrophage RAW 264.7 cells were transfected with the phRL-TK reporter gene and the NF-κB luciferase construct as described in Materials and methods, together with pcDNA3.1 or 100 ng A46R. Cells were stimulated for 6 h with 10 nM MALP-2 (MALP), 5 μg/ml Pam3Cys (Pam), 1 μg/ml LPS, 250 ng/ml Flagellin (Flag), 1 μM R-848, or 5 μg/ml CpG DNA. Cells were harvested 24 h after transfection and the reporter gene activity was measured.

    Journal: The Journal of Experimental Medicine

    Article Title: Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

    doi: 10.1084/jem.20041442

    Figure Lengend Snippet: A46R associates with MyD88 and blocks MyD88-dependent signaling. (a) Alignment of A46R with human TIR domains. The conserved motifs Box 1, Box 2, and Box 3 are indicated by a solid line. The eight differences between VV and variola virus A46R sequences are indicated by an asterisk. For A46R, amino acids 35–238 are shown. (b) HEK 293T cells were transfected with A46R and AU1-MyD88 (left) or Flag-TRAF2 (right) as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (c) HEK 293T cells were transfected with 8 μg AU1-MyD88 (top) or Flag-TRAF2 (bottom). After 24 h, lysates were incubated with GST alone (lane 2) or GST-A46R (lane 3), and together with whole cell lysates (lane 1) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (d) HEK 293 cells were transfected with 4 μg of myc-MyD88. After 24 h, cells were infected with viruses either containing (vWT-A46R) or not (vΔA46R) the A46R gene (MOI = 1) and harvested 24 h after infection. Lysates were subjected to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. Whole cell lysates were analyzed for expression of A46R. (e) Murine macrophage RAW 264.7 cells were transfected with the phRL-TK reporter gene and the NF-κB luciferase construct as described in Materials and methods, together with pcDNA3.1 or 100 ng A46R. Cells were stimulated for 6 h with 10 nM MALP-2 (MALP), 5 μg/ml Pam3Cys (Pam), 1 μg/ml LPS, 250 ng/ml Flagellin (Flag), 1 μM R-848, or 5 μg/ml CpG DNA. Cells were harvested 24 h after transfection and the reporter gene activity was measured.

    Article Snippet: The total amount of DNA per transfection was kept constant at 220 ng (HEK293) or 200 ng (RAW264.7) by addition of pcDNA3.1 (Stratagene).

    Techniques: Transfection, Immunoprecipitation, SDS Page, Incubation, Infection, Expressing, Luciferase, Construct, Activity Assay

    A46R inhibits TLR4 signaling and interacts with TLR4, Mal, and TRAM. (a) HEK 293 cells were transfected with 50 ng CD4-TLR4, 25–100 ng A46R, or pcDNA3.1 (EV) and the NF-κB (left), p38 (middle), or ERK (right) reporter plasmids as indicated. Cells were harvested 24 h after transfection, and luciferase reporter gene activity was measured. (b–d) HEK 293T cells were transfected with A46R and Flag-TLR4 (b), Flag-Mal (c), or Flag-TRAM (d) as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (e) HEK 293T cells were transfected with 8 μg of Flag-TLR4 (top), Flag-Mal (middle), or Flag-TRAM (bottom). After 24 h, lysates were incubated with GST-A46R (lane 3) or GST alone (lane 2), and together with whole cell lysate (lane 1), were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (f) HEK-TLR4 cells were transfected with the indicated amounts (ng) of A46R 24 h before stimulation with 1 μg/ml LPS, and 24 h after stimulation, supernatants were harvested and assayed for IL-8 by ELISA.

    Journal: The Journal of Experimental Medicine

    Article Title: Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

    doi: 10.1084/jem.20041442

    Figure Lengend Snippet: A46R inhibits TLR4 signaling and interacts with TLR4, Mal, and TRAM. (a) HEK 293 cells were transfected with 50 ng CD4-TLR4, 25–100 ng A46R, or pcDNA3.1 (EV) and the NF-κB (left), p38 (middle), or ERK (right) reporter plasmids as indicated. Cells were harvested 24 h after transfection, and luciferase reporter gene activity was measured. (b–d) HEK 293T cells were transfected with A46R and Flag-TLR4 (b), Flag-Mal (c), or Flag-TRAM (d) as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (e) HEK 293T cells were transfected with 8 μg of Flag-TLR4 (top), Flag-Mal (middle), or Flag-TRAM (bottom). After 24 h, lysates were incubated with GST-A46R (lane 3) or GST alone (lane 2), and together with whole cell lysate (lane 1), were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (f) HEK-TLR4 cells were transfected with the indicated amounts (ng) of A46R 24 h before stimulation with 1 μg/ml LPS, and 24 h after stimulation, supernatants were harvested and assayed for IL-8 by ELISA.

    Article Snippet: The total amount of DNA per transfection was kept constant at 220 ng (HEK293) or 200 ng (RAW264.7) by addition of pcDNA3.1 (Stratagene).

    Techniques: Transfection, Luciferase, Activity Assay, Immunoprecipitation, SDS Page, Incubation, Enzyme-linked Immunosorbent Assay

    A46R associates with TRIF and inhibits TRIF-dependent signaling and gene induction. (a) HEK 293T cells were transfected with A46R and Flag-TRIF as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (b) HEK 293T cells were transfected with 8 μg Flag-TRIF. After 24 h, lysates were incubated with GST-A46R (lane 3) or GST alone (lane 2), and together with whole cell lysates (lane 1), were analyzed by SDS-PAGE and immunoblotting with anti-Flag Ab. (c) HEK 293 cells were transfected with the IRF3 reporter plasmids (as described in Materials and methods) with either 50 ng CD4-TLR4 (top) or 0.5 ng TLR3 (bottom) and 50–150 ng A46R or pcDNA3.1 (EV) as indicated. (bottom) Cells were stimulated with 25 μg/ml poly(I:C) 6 h before harvesting. Luciferase activity was measured after 24 h. (d) HEK-TLR3 cells were transfected with the indicated amounts (ng) of A46R 24 h before stimulation with 25 μg/ml poly(I:C), and 24 h after stimulation supernatants were harvested and assayed for RANTES by ELISA. (e and f) As in a and b, except Flag-SARM was transfected instead of Flag-TRIF.

    Journal: The Journal of Experimental Medicine

    Article Title: Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

    doi: 10.1084/jem.20041442

    Figure Lengend Snippet: A46R associates with TRIF and inhibits TRIF-dependent signaling and gene induction. (a) HEK 293T cells were transfected with A46R and Flag-TRIF as indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. (b) HEK 293T cells were transfected with 8 μg Flag-TRIF. After 24 h, lysates were incubated with GST-A46R (lane 3) or GST alone (lane 2), and together with whole cell lysates (lane 1), were analyzed by SDS-PAGE and immunoblotting with anti-Flag Ab. (c) HEK 293 cells were transfected with the IRF3 reporter plasmids (as described in Materials and methods) with either 50 ng CD4-TLR4 (top) or 0.5 ng TLR3 (bottom) and 50–150 ng A46R or pcDNA3.1 (EV) as indicated. (bottom) Cells were stimulated with 25 μg/ml poly(I:C) 6 h before harvesting. Luciferase activity was measured after 24 h. (d) HEK-TLR3 cells were transfected with the indicated amounts (ng) of A46R 24 h before stimulation with 25 μg/ml poly(I:C), and 24 h after stimulation supernatants were harvested and assayed for RANTES by ELISA. (e and f) As in a and b, except Flag-SARM was transfected instead of Flag-TRIF.

    Article Snippet: The total amount of DNA per transfection was kept constant at 220 ng (HEK293) or 200 ng (RAW264.7) by addition of pcDNA3.1 (Stratagene).

    Techniques: Transfection, Immunoprecipitation, SDS Page, Incubation, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay

    A46R inhibits multiple IL-1–dependent signals. HEK 293 cells were transfected with 100 ng A46R or pcDNA3.1 (EV) and the NF-κB (a), p65 (b), JNK (c), or ERK (d) reporter plasmids as described in Materials and methods. 6 h before harvesting, the cells were stimulated with either 100 ng/ml IL-1 or 100 ng/ml TNF as indicated, and luciferase reporter gene activity was measured.

    Journal: The Journal of Experimental Medicine

    Article Title: Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

    doi: 10.1084/jem.20041442

    Figure Lengend Snippet: A46R inhibits multiple IL-1–dependent signals. HEK 293 cells were transfected with 100 ng A46R or pcDNA3.1 (EV) and the NF-κB (a), p65 (b), JNK (c), or ERK (d) reporter plasmids as described in Materials and methods. 6 h before harvesting, the cells were stimulated with either 100 ng/ml IL-1 or 100 ng/ml TNF as indicated, and luciferase reporter gene activity was measured.

    Article Snippet: The total amount of DNA per transfection was kept constant at 220 ng (HEK293) or 200 ng (RAW264.7) by addition of pcDNA3.1 (Stratagene).

    Techniques: Transfection, Luciferase, Activity Assay

    NP expression in mammalian cells leads to downreglation of PKR and eIF2α phosphorylation. HEK293T cells were transfected with NP expressing plasmid (pcDNA3.1-NP) or control plasmid (pcDNA3.1). Cells were harvested at 36 hours post-transfection and cell lysates were subjected to western blotting analysis. A. Lane 1 of panel 2 shows significant downregulation of pPKR levels in NP transfected cells. Panel 1 shows NP expression level, whereas panel 3 shows equal loading trough β-Actin control. B. Figure shows graphical representation of relative pPKR levels as measured by western blotting followed by densitometric measurement in 3 independent experiments. Error bars represent standard deviation. C. Lane 1 of panel 2 shows significant downregulation of p-eIF2α levels in NP transfected cells. Panel 1 shows NP expression level, whereas panel 3 shows equal loading trough β-Actin control. D. Figure shows graphical representation of relative p-eIF2α levels as measured by western blotting followed by densitometric measurement in 3 independent experiments. Error bars represent standard deviation.

    Journal: PLoS ONE

    Article Title: Influenza A Virus Nucleoprotein Exploits Hsp40 to Inhibit PKR Activation

    doi: 10.1371/journal.pone.0020215

    Figure Lengend Snippet: NP expression in mammalian cells leads to downreglation of PKR and eIF2α phosphorylation. HEK293T cells were transfected with NP expressing plasmid (pcDNA3.1-NP) or control plasmid (pcDNA3.1). Cells were harvested at 36 hours post-transfection and cell lysates were subjected to western blotting analysis. A. Lane 1 of panel 2 shows significant downregulation of pPKR levels in NP transfected cells. Panel 1 shows NP expression level, whereas panel 3 shows equal loading trough β-Actin control. B. Figure shows graphical representation of relative pPKR levels as measured by western blotting followed by densitometric measurement in 3 independent experiments. Error bars represent standard deviation. C. Lane 1 of panel 2 shows significant downregulation of p-eIF2α levels in NP transfected cells. Panel 1 shows NP expression level, whereas panel 3 shows equal loading trough β-Actin control. D. Figure shows graphical representation of relative p-eIF2α levels as measured by western blotting followed by densitometric measurement in 3 independent experiments. Error bars represent standard deviation.

    Article Snippet: NP gene of A/ Hatay/ 2004 (H5N1) influenza virus was cloned in pCDNA3.1 myc his vector (Invitrogen), pEGFPN1 vector (Clontech) and pGBKT7 vectors (Clontech) to generate myc-tagged, GFP-tagged and GAL4 DNA BD fused NP, respectively.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Standard Deviation

    Co-localization of IAV NP and Hsp40 in nucleus of mammalian cells. A and B. A549 cells were transfected with pcDNA3.1-NP or control pcDNA3.1 plasmid for 24 hours, and cells were fixed and processed for immunostaining. NP was stained using anti-Myc tag specific primary antibody and Alexa488 conjugated secondary antibody (Green). Hsp40 was stained using Hsp40 specific primary antibody and Alexa 594 conjugated secondary antibody (Red). Nuclei were stained with DAPI. A shows pcDNA3.1-NP transfected cells whereas B shows control pcDNA3.1 transfected cells. Panels are labeled for their respective staining. Lower right panel shows nuclear colocalization of NP and Hsp40. C and D. A549 cells were infected with PR8 influenza A virus at 1 MOI for 24 hours, and cells were fixed and processed for immunostaining. NP was stained using anti-NP monoclonal primary antibody and Alexa488 conjugated secondary antibody (Green). Hsp40 was stained using Hsp40 specific primary antibody and Alexa 594 conjugated secondary antibody (Red). Nuclei were stained with DAPI. Panels are labeled for their respective staining. C shows PR8 infected cells whereas D shows control uninfected cells. Lower right panel in C shows primarily nuclear colocalization of NP and Hsp40.

    Journal: PLoS ONE

    Article Title: Influenza A Virus Nucleoprotein Exploits Hsp40 to Inhibit PKR Activation

    doi: 10.1371/journal.pone.0020215

    Figure Lengend Snippet: Co-localization of IAV NP and Hsp40 in nucleus of mammalian cells. A and B. A549 cells were transfected with pcDNA3.1-NP or control pcDNA3.1 plasmid for 24 hours, and cells were fixed and processed for immunostaining. NP was stained using anti-Myc tag specific primary antibody and Alexa488 conjugated secondary antibody (Green). Hsp40 was stained using Hsp40 specific primary antibody and Alexa 594 conjugated secondary antibody (Red). Nuclei were stained with DAPI. A shows pcDNA3.1-NP transfected cells whereas B shows control pcDNA3.1 transfected cells. Panels are labeled for their respective staining. Lower right panel shows nuclear colocalization of NP and Hsp40. C and D. A549 cells were infected with PR8 influenza A virus at 1 MOI for 24 hours, and cells were fixed and processed for immunostaining. NP was stained using anti-NP monoclonal primary antibody and Alexa488 conjugated secondary antibody (Green). Hsp40 was stained using Hsp40 specific primary antibody and Alexa 594 conjugated secondary antibody (Red). Nuclei were stained with DAPI. Panels are labeled for their respective staining. C shows PR8 infected cells whereas D shows control uninfected cells. Lower right panel in C shows primarily nuclear colocalization of NP and Hsp40.

    Article Snippet: NP gene of A/ Hatay/ 2004 (H5N1) influenza virus was cloned in pCDNA3.1 myc his vector (Invitrogen), pEGFPN1 vector (Clontech) and pGBKT7 vectors (Clontech) to generate myc-tagged, GFP-tagged and GAL4 DNA BD fused NP, respectively.

    Techniques: Transfection, Plasmid Preparation, Immunostaining, Staining, Labeling, Infection

    Detection of IAV NP-Hsp40 interaction in mammalian cells tranfected with NP expressing plasmid by co-immunoprecipitation. A. HEK293T cells were pcDNA3.1-NP and pcDNA3.1-Hsp40 plasmids alone or in combination, followed by metabolic labeling with S 35 . 48 hours post-transfection cells were harvested and IP was setup using anti-NP-specific antibody and anti-Hsp40-specific antibody followed by autoradiography. Lanes 2 and 4 show co-IP of Hsp40 with NP and vice-versa. Lanes 1 and 3 show anti-NP and anti-Hsp40 antibodies were not cross reacting with Hsp40 and NP, respectively. B. A549 cells were transfected with pcDNA3.1-NP plasmid or control pcDNA3.1 plasmid. Cells were harvested 48 hours post-transfection and immunoprecipitation was setup using anti-Myc tag antibody and anti-Hsp40 antibody, followed by western blotting. Lane 2 of panel 1 shows co-IP of NP with Hsp40 and lane 2 of panel 2 shows co-IP Hsp40 with NP. Lane 1 of panel 1 and 2 represents control samples transfected with empty vector. Panels 3 and 4 show expression levels of NP and Hsp40 in cell lysates.

    Journal: PLoS ONE

    Article Title: Influenza A Virus Nucleoprotein Exploits Hsp40 to Inhibit PKR Activation

    doi: 10.1371/journal.pone.0020215

    Figure Lengend Snippet: Detection of IAV NP-Hsp40 interaction in mammalian cells tranfected with NP expressing plasmid by co-immunoprecipitation. A. HEK293T cells were pcDNA3.1-NP and pcDNA3.1-Hsp40 plasmids alone or in combination, followed by metabolic labeling with S 35 . 48 hours post-transfection cells were harvested and IP was setup using anti-NP-specific antibody and anti-Hsp40-specific antibody followed by autoradiography. Lanes 2 and 4 show co-IP of Hsp40 with NP and vice-versa. Lanes 1 and 3 show anti-NP and anti-Hsp40 antibodies were not cross reacting with Hsp40 and NP, respectively. B. A549 cells were transfected with pcDNA3.1-NP plasmid or control pcDNA3.1 plasmid. Cells were harvested 48 hours post-transfection and immunoprecipitation was setup using anti-Myc tag antibody and anti-Hsp40 antibody, followed by western blotting. Lane 2 of panel 1 shows co-IP of NP with Hsp40 and lane 2 of panel 2 shows co-IP Hsp40 with NP. Lane 1 of panel 1 and 2 represents control samples transfected with empty vector. Panels 3 and 4 show expression levels of NP and Hsp40 in cell lysates.

    Article Snippet: NP gene of A/ Hatay/ 2004 (H5N1) influenza virus was cloned in pCDNA3.1 myc his vector (Invitrogen), pEGFPN1 vector (Clontech) and pGBKT7 vectors (Clontech) to generate myc-tagged, GFP-tagged and GAL4 DNA BD fused NP, respectively.

    Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Labeling, Transfection, Autoradiography, Co-Immunoprecipitation Assay, Western Blot