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  • 99
    Thermo Fisher pcdna3 1
    TESC mediates the activation of the IGF1R/c-Src/STAT3 signaling pathway. ( A ) The activation of c-Src, FAK, and STAT3 was assayed using western blot analysis in A549 cells transfected with the siRNA and H460 cells transfected with TESC <t>-pcDNA3.1</t> vector. ( B ) The interactions between TESC, c-Src and IGF1Rβ were determined by immunoprecipitation assays in A549 cells. ( C ) The interactions between TESC, JAK2 and FAK were evaluated by immunoprecipitation assays in A549 cells. ( D ) Phosphorylation levels of IGF1Rβ in c - Src knockdown A549 cells and c - Src -overexpressing H460 cells. ( E ) Fluorescence-activated cell sorting (FACS) analysis of ALDH1 in c - Src knockdown A549 cells and c - Src -overexpressing H460 cells was performed using ALDEFLUOR assay. ( F ) Colony forming, sphere forming and metastatic analysis in c - Src knockdown A549 cells. Data represent mean ± SD of three independent experiments using two-tailed t-test. *P
    Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 52083 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna3 1 expression vector
    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 <t>pcDNA3</t> (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
    Pcdna3 1 Expression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4924 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pcdna
    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 <t>pcDNA3</t> (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
    Pcdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5297 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pcdna - by Bioz Stars, 2020-11
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    99
    Thermo Fisher pcdna5 frt to vector
    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 <t>pcDNA3</t> (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
    Pcdna5 Frt To Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1923 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna5 frt vector
    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 <t>pcDNA3</t> (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
    Pcdna5 Frt Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 893 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pcdna3 1 zeo
    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 <t>pcDNA3</t> (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
    Pcdna3 1 Zeo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 859 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pcdna3 1 hygro
    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 <t>pcDNA3</t> (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
    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
    https://www.bioz.com/result/pcdna3 1 hygro/product/Thermo Fisher
    Average 99 stars, based on 902 article reviews
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    pcdna3 1 hygro - by Bioz Stars, 2020-11
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    99
    Thermo Fisher pcdna3 1zeo
    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 <t>pcDNA3</t> (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
    Pcdna3 1zeo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 460 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1zeo/product/Thermo Fisher
    Average 99 stars, based on 460 article reviews
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    pcdna3 1zeo - by Bioz Stars, 2020-11
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    Image Search Results


    TESC mediates the activation of the IGF1R/c-Src/STAT3 signaling pathway. ( A ) The activation of c-Src, FAK, and STAT3 was assayed using western blot analysis in A549 cells transfected with the siRNA and H460 cells transfected with TESC -pcDNA3.1 vector. ( B ) The interactions between TESC, c-Src and IGF1Rβ were determined by immunoprecipitation assays in A549 cells. ( C ) The interactions between TESC, JAK2 and FAK were evaluated by immunoprecipitation assays in A549 cells. ( D ) Phosphorylation levels of IGF1Rβ in c - Src knockdown A549 cells and c - Src -overexpressing H460 cells. ( E ) Fluorescence-activated cell sorting (FACS) analysis of ALDH1 in c - Src knockdown A549 cells and c - Src -overexpressing H460 cells was performed using ALDEFLUOR assay. ( F ) Colony forming, sphere forming and metastatic analysis in c - Src knockdown A549 cells. Data represent mean ± SD of three independent experiments using two-tailed t-test. *P

    Journal: Scientific Reports

    Article Title: Tescalcin/c-Src/IGF1Rβ-mediated STAT3 activation enhances cancer stemness and radioresistant properties through ALDH1

    doi: 10.1038/s41598-018-29142-x

    Figure Lengend Snippet: TESC mediates the activation of the IGF1R/c-Src/STAT3 signaling pathway. ( A ) The activation of c-Src, FAK, and STAT3 was assayed using western blot analysis in A549 cells transfected with the siRNA and H460 cells transfected with TESC -pcDNA3.1 vector. ( B ) The interactions between TESC, c-Src and IGF1Rβ were determined by immunoprecipitation assays in A549 cells. ( C ) The interactions between TESC, JAK2 and FAK were evaluated by immunoprecipitation assays in A549 cells. ( D ) Phosphorylation levels of IGF1Rβ in c - Src knockdown A549 cells and c - Src -overexpressing H460 cells. ( E ) Fluorescence-activated cell sorting (FACS) analysis of ALDH1 in c - Src knockdown A549 cells and c - Src -overexpressing H460 cells was performed using ALDEFLUOR assay. ( F ) Colony forming, sphere forming and metastatic analysis in c - Src knockdown A549 cells. Data represent mean ± SD of three independent experiments using two-tailed t-test. *P

    Article Snippet: Cells were transiently transfected with pcDNA3.1(+) expression vectors using Lipofectamine 2000 (Invitrogen).

    Techniques: Activation Assay, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Fluorescence, FACS, Two Tailed Test

    TESC regulates the CSC-like self-renewal properties in lung cancer cells. ( A ) Clonogenicities of TESC -knockdown A549 cells with siRNA and TESC -overexpressing H460 cells transfected with expression vector ( TESC -pcDNA3.1) were examined in vitro by colony formation assay. ( B ) Cellular levels of TESC and CSC markers including CD44, CD133, Oct3/4, Sox2, and β-catenin in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( C ) RT-PCR analysis of ALDH1 isozymes in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( D ) Changes in sphere-forming capacity in TESC -knockdown A549 cells or TESC -overexpressing H460 cells. ( E ) Changes in cellular ALDH1 levels were examined using ALDEFLUOR assay in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. Data represent mean ± SD of three independent experiments using two-tailed t-test. *P

    Journal: Scientific Reports

    Article Title: Tescalcin/c-Src/IGF1Rβ-mediated STAT3 activation enhances cancer stemness and radioresistant properties through ALDH1

    doi: 10.1038/s41598-018-29142-x

    Figure Lengend Snippet: TESC regulates the CSC-like self-renewal properties in lung cancer cells. ( A ) Clonogenicities of TESC -knockdown A549 cells with siRNA and TESC -overexpressing H460 cells transfected with expression vector ( TESC -pcDNA3.1) were examined in vitro by colony formation assay. ( B ) Cellular levels of TESC and CSC markers including CD44, CD133, Oct3/4, Sox2, and β-catenin in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( C ) RT-PCR analysis of ALDH1 isozymes in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( D ) Changes in sphere-forming capacity in TESC -knockdown A549 cells or TESC -overexpressing H460 cells. ( E ) Changes in cellular ALDH1 levels were examined using ALDEFLUOR assay in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. Data represent mean ± SD of three independent experiments using two-tailed t-test. *P

    Article Snippet: Cells were transiently transfected with pcDNA3.1(+) expression vectors using Lipofectamine 2000 (Invitrogen).

    Techniques: Transfection, Expressing, Plasmid Preparation, In Vitro, Colony Assay, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    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

    Over-expression and silencing of p53 produce reciprocal effect on the activation pattern of NF-κB, MAPKs and AP-1 . (A-B) H1299 and A549 cells were transfected with pcDNA3-p53 WT and pcDNA3-p53 DN constructs respectively and the stable clones selected were lysed and Western blotted against p53 antibody. (C-D) H1299-p53 WT and A549-p53 DN cells were treated with nicotine (10 -9 -10 -2 M) for 30 min, and EMSA was done using nuclear extracts. (E) A549 cells were treated with different concentrations of pifithrin-α (10-50 μM) for 4 h and the whole cell lyasates prepared were Western blotted against p53 antibody. (F) A549 cells were treated with 40 μM pifithrin-α for 4 h followed by nicotine (10 -9 -10 -2 M) for 30 min and EMSA was done. (G-H) H1299-p53 WT and A549-p53 DN cells were treated with nicotine (10 -9 -10 -2 M) for 30 min, and EMSA was done. (I-J) H1299 WT-p53 and A549 DN-p53 cells were treated with nicotine (10 -9 -10 -2 M) for 30 min and whole cell lysates were Western blotted against respective phospho specific antibodies. All blots and EMSAs are representative samples of three independent experiments.

    Journal: Molecular Cancer

    Article Title: Nicotine-induced survival signaling in lung cancer cells is dependent on their p53 status while its down-regulation by curcumin is independent

    doi: 10.1186/1476-4598-9-220

    Figure Lengend Snippet: Over-expression and silencing of p53 produce reciprocal effect on the activation pattern of NF-κB, MAPKs and AP-1 . (A-B) H1299 and A549 cells were transfected with pcDNA3-p53 WT and pcDNA3-p53 DN constructs respectively and the stable clones selected were lysed and Western blotted against p53 antibody. (C-D) H1299-p53 WT and A549-p53 DN cells were treated with nicotine (10 -9 -10 -2 M) for 30 min, and EMSA was done using nuclear extracts. (E) A549 cells were treated with different concentrations of pifithrin-α (10-50 μM) for 4 h and the whole cell lyasates prepared were Western blotted against p53 antibody. (F) A549 cells were treated with 40 μM pifithrin-α for 4 h followed by nicotine (10 -9 -10 -2 M) for 30 min and EMSA was done. (G-H) H1299-p53 WT and A549-p53 DN cells were treated with nicotine (10 -9 -10 -2 M) for 30 min, and EMSA was done. (I-J) H1299 WT-p53 and A549 DN-p53 cells were treated with nicotine (10 -9 -10 -2 M) for 30 min and whole cell lysates were Western blotted against respective phospho specific antibodies. All blots and EMSAs are representative samples of three independent experiments.

    Article Snippet: Stable Transfection H1299 Cells were transfected with pcDNA3-p53WT or the empty vector pcDNA3 while A549 cells were transfected with pcDNA3-p53 DN or the empty vector pcDNA3 using the calcium-phosphate transfection kit (Life Technologies, Inc.) according to manufacturer's protocol and selected using G418 (150 μg/ml) [ ].

    Techniques: Over Expression, Activation Assay, Transfection, Construct, Clone Assay, Western Blot