anti egr1 15f7 cs 4351  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti egr1 15f7 cs 4351
    HGF-induced activation of <t>Egr1</t> mRNA and protein level ( 2A, 2C ) and the effect of heparin on the HGF induced Egr1 expression in SK-HEP-1 cells were examined by RT-PCR and western blotting ( 2B, 2D ). Egr1 transcriptional activity was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with pGL2-Luc-B-Egr1 plasmid construct and the pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity ( 2E, 2F ). Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.
    Anti Egr1 15f7 Cs 4351, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti egr1 15f7 cs 4351/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti egr1 15f7 cs 4351 - by Bioz Stars, 2023-02
    95/100 stars

    Images

    1) Product Images from "Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1"

    Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042717

    HGF-induced activation of Egr1 mRNA and protein level ( 2A, 2C ) and the effect of heparin on the HGF induced Egr1 expression in SK-HEP-1 cells were examined by RT-PCR and western blotting ( 2B, 2D ). Egr1 transcriptional activity was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with pGL2-Luc-B-Egr1 plasmid construct and the pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity ( 2E, 2F ). Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.
    Figure Legend Snippet: HGF-induced activation of Egr1 mRNA and protein level ( 2A, 2C ) and the effect of heparin on the HGF induced Egr1 expression in SK-HEP-1 cells were examined by RT-PCR and western blotting ( 2B, 2D ). Egr1 transcriptional activity was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with pGL2-Luc-B-Egr1 plasmid construct and the pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity ( 2E, 2F ). Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

    Techniques Used: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Construct

    HGF-induced activation of Egr1 and the effect of c-Met inhibition on HGF-induced Egr1 expression in SK-HEP-1 cells were examined by immunoblotting and luciferase reporter assays ( 5A, 5B ). Transcriptional activity of Egr1 was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with a pGL2-Luc-B-Egr1 plasmid construct and pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity. Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.
    Figure Legend Snippet: HGF-induced activation of Egr1 and the effect of c-Met inhibition on HGF-induced Egr1 expression in SK-HEP-1 cells were examined by immunoblotting and luciferase reporter assays ( 5A, 5B ). Transcriptional activity of Egr1 was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with a pGL2-Luc-B-Egr1 plasmid construct and pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity. Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

    Techniques Used: Activation Assay, Inhibition, Expressing, Western Blot, Luciferase, Activity Assay, Reporter Assay, Transfection, Plasmid Preparation, Construct

    To achieve an inducible knockdown of Egr1, we firstly transfected SK-HEP-1 cells with pCDNA6/TR which express tet repressor. Stable cells were selected using Blasticidin before co-transfecting SK-HEP-1 T-REx cells with tetracycline–regulated pSUPER.retro.neo+GFP tet RNAi construct, which was directed against a sequence containing Egr1. Stable cells were selected using G418. After selection, clones were analyzed for downregulation of Egr1 after 24 h of tetracycline treatment. Ten-fold down-regulation of Egr1 was detected by immunoblotting in pSUPER.retro.neo+GFP tet/Egr1 clones ( 6A ) but not in mock transfected clones ( 6B ). The graph compares the signal intensities obtained from Egr1 bands.
    Figure Legend Snippet: To achieve an inducible knockdown of Egr1, we firstly transfected SK-HEP-1 cells with pCDNA6/TR which express tet repressor. Stable cells were selected using Blasticidin before co-transfecting SK-HEP-1 T-REx cells with tetracycline–regulated pSUPER.retro.neo+GFP tet RNAi construct, which was directed against a sequence containing Egr1. Stable cells were selected using G418. After selection, clones were analyzed for downregulation of Egr1 after 24 h of tetracycline treatment. Ten-fold down-regulation of Egr1 was detected by immunoblotting in pSUPER.retro.neo+GFP tet/Egr1 clones ( 6A ) but not in mock transfected clones ( 6B ). The graph compares the signal intensities obtained from Egr1 bands.

    Techniques Used: Transfection, Construct, Sequencing, Selection, Clone Assay, Western Blot

    SK-HEP-1 TREx cells were obtained by stable transfection of pCDNA-6/TR. Then SK-HEP-1 TREx cells were transfected with recombinant pSUPER-retro.neo+GFP vector expressing Egr-1 targeted shRNA or empty pSUPER-retro.neo+GFP vector using Fugene as described in . Knockdown of Egr1 in Egr1-shRNA-expressing SK-HEP-1 stable cell lines were induced by tetracycline ( 7A ). Equal amount of tetracycline induced and un-induced Egr1-shRNA-expressing SK-HEP-1 cells were used for migration and invasion assays in the presence and absence of HGF ( 7B, 7C ). Results are expressed as fold differences of matrigel invaded cells and are representative of two or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups. Zymographic gel showing active MMP-2 and-9 bands in 24 h conditioned medium of cultured Egr1-shRNA-expressing SK-HEP-1 cells left untreated or stimulated with tetracycline and/or HGF ( 7D, 7E ). Protein levels of MT1-MMP were studied in tetracyclin and/or HGF-induced SK-HEP-1-Egr1 cells ( 7F ). Densitometric analysis of gelatin zymography and immunoblots showed that HGF induces MT1-MMP expression and MMP-2 and -9 activation, whereas silencing of Egr1 blocks HGF-induced MMPs up-regulation.
    Figure Legend Snippet: SK-HEP-1 TREx cells were obtained by stable transfection of pCDNA-6/TR. Then SK-HEP-1 TREx cells were transfected with recombinant pSUPER-retro.neo+GFP vector expressing Egr-1 targeted shRNA or empty pSUPER-retro.neo+GFP vector using Fugene as described in . Knockdown of Egr1 in Egr1-shRNA-expressing SK-HEP-1 stable cell lines were induced by tetracycline ( 7A ). Equal amount of tetracycline induced and un-induced Egr1-shRNA-expressing SK-HEP-1 cells were used for migration and invasion assays in the presence and absence of HGF ( 7B, 7C ). Results are expressed as fold differences of matrigel invaded cells and are representative of two or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups. Zymographic gel showing active MMP-2 and-9 bands in 24 h conditioned medium of cultured Egr1-shRNA-expressing SK-HEP-1 cells left untreated or stimulated with tetracycline and/or HGF ( 7D, 7E ). Protein levels of MT1-MMP were studied in tetracyclin and/or HGF-induced SK-HEP-1-Egr1 cells ( 7F ). Densitometric analysis of gelatin zymography and immunoblots showed that HGF induces MT1-MMP expression and MMP-2 and -9 activation, whereas silencing of Egr1 blocks HGF-induced MMPs up-regulation.

    Techniques Used: Stable Transfection, Transfection, Recombinant, Plasmid Preparation, Expressing, shRNA, Migration, Cell Culture, Zymography, Western Blot, Activation Assay

    The effect of Egr1 on the cell invasion of HCC cell lines were examined using SNU-449 transiently transfected with pCMV-6-AC-GFP Egr1 overexpression vector ( 8A ). Equal amounts of wild type and Egr1 overexpressing SNU-449 cells were used for migration ( 8B ) and invasion ( 8C ) assay in the presence and absence of HGF. Results are expressed as fold differences of matrigel invaded cells. Zymographic gel showing the active MMP-9 bands in 24 h conditioned medium of cultured Egr1-overexpressing SNU-449 cells ( 8D ). The graph compares the signal intensities obtained from zymography gels.
    Figure Legend Snippet: The effect of Egr1 on the cell invasion of HCC cell lines were examined using SNU-449 transiently transfected with pCMV-6-AC-GFP Egr1 overexpression vector ( 8A ). Equal amounts of wild type and Egr1 overexpressing SNU-449 cells were used for migration ( 8B ) and invasion ( 8C ) assay in the presence and absence of HGF. Results are expressed as fold differences of matrigel invaded cells. Zymographic gel showing the active MMP-9 bands in 24 h conditioned medium of cultured Egr1-overexpressing SNU-449 cells ( 8D ). The graph compares the signal intensities obtained from zymography gels.

    Techniques Used: Transfection, Over Expression, Plasmid Preparation, Migration, Cell Culture, Zymography

    The effects of heparin and HGF on Egr1 expression in Egr1 over-expressing SNU-449 cells was analyzed by immunoblotting ( 9A ). Following serum deprivation, Egr1 overexpressed SN-449 cells were left untreated (0) or treated with HGF in the presence or absence of heparin, then invasion and migration assays were performed using the Roche xCELLigence System ( 9B, 9C ). Results are representative of three or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.
    Figure Legend Snippet: The effects of heparin and HGF on Egr1 expression in Egr1 over-expressing SNU-449 cells was analyzed by immunoblotting ( 9A ). Following serum deprivation, Egr1 overexpressed SN-449 cells were left untreated (0) or treated with HGF in the presence or absence of heparin, then invasion and migration assays were performed using the Roche xCELLigence System ( 9B, 9C ). Results are representative of three or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

    Techniques Used: Expressing, Western Blot, Migration

    Activation of HGF/c-Met signaling in HCC cells leads to MAPK signaling (i), and up-regulation of Egr1 (ii), which in turn induces MMP-2 and MMP-9 activation and MT1-MMP expression (iii), and increased cell motility and invasion (iv). Heparin treatment results in the inhibition of HGF-induced cellular invasion via repression of HGF-induced c-Met activation, as well as inhibition of MAPK signaling, downregulation of Egr1 expression, and MMP activation (v).
    Figure Legend Snippet: Activation of HGF/c-Met signaling in HCC cells leads to MAPK signaling (i), and up-regulation of Egr1 (ii), which in turn induces MMP-2 and MMP-9 activation and MT1-MMP expression (iii), and increased cell motility and invasion (iv). Heparin treatment results in the inhibition of HGF-induced cellular invasion via repression of HGF-induced c-Met activation, as well as inhibition of MAPK signaling, downregulation of Egr1 expression, and MMP activation (v).

    Techniques Used: Activation Assay, Expressing, Inhibition

    anti egr1 15f7 cs 4351  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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  • 95

    Structured Review

    Cell Signaling Technology Inc anti egr1 15f7 cs 4351
    HGF-induced activation of <t>Egr1</t> mRNA and protein level ( 2A, 2C ) and the effect of heparin on the HGF induced Egr1 expression in SK-HEP-1 cells were examined by RT-PCR and western blotting ( 2B, 2D ). Egr1 transcriptional activity was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with pGL2-Luc-B-Egr1 plasmid construct and the pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity ( 2E, 2F ). Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.
    Anti Egr1 15f7 Cs 4351, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti egr1 15f7 cs 4351/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti egr1 15f7 cs 4351 - by Bioz Stars, 2023-02
    95/100 stars

    Images

    1) Product Images from "Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1"

    Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042717

    HGF-induced activation of Egr1 mRNA and protein level ( 2A, 2C ) and the effect of heparin on the HGF induced Egr1 expression in SK-HEP-1 cells were examined by RT-PCR and western blotting ( 2B, 2D ). Egr1 transcriptional activity was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with pGL2-Luc-B-Egr1 plasmid construct and the pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity ( 2E, 2F ). Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.
    Figure Legend Snippet: HGF-induced activation of Egr1 mRNA and protein level ( 2A, 2C ) and the effect of heparin on the HGF induced Egr1 expression in SK-HEP-1 cells were examined by RT-PCR and western blotting ( 2B, 2D ). Egr1 transcriptional activity was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with pGL2-Luc-B-Egr1 plasmid construct and the pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity ( 2E, 2F ). Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

    Techniques Used: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Construct

    HGF-induced activation of Egr1 and the effect of c-Met inhibition on HGF-induced Egr1 expression in SK-HEP-1 cells were examined by immunoblotting and luciferase reporter assays ( 5A, 5B ). Transcriptional activity of Egr1 was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with a pGL2-Luc-B-Egr1 plasmid construct and pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity. Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.
    Figure Legend Snippet: HGF-induced activation of Egr1 and the effect of c-Met inhibition on HGF-induced Egr1 expression in SK-HEP-1 cells were examined by immunoblotting and luciferase reporter assays ( 5A, 5B ). Transcriptional activity of Egr1 was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with a pGL2-Luc-B-Egr1 plasmid construct and pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity. Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

    Techniques Used: Activation Assay, Inhibition, Expressing, Western Blot, Luciferase, Activity Assay, Reporter Assay, Transfection, Plasmid Preparation, Construct

    To achieve an inducible knockdown of Egr1, we firstly transfected SK-HEP-1 cells with pCDNA6/TR which express tet repressor. Stable cells were selected using Blasticidin before co-transfecting SK-HEP-1 T-REx cells with tetracycline–regulated pSUPER.retro.neo+GFP tet RNAi construct, which was directed against a sequence containing Egr1. Stable cells were selected using G418. After selection, clones were analyzed for downregulation of Egr1 after 24 h of tetracycline treatment. Ten-fold down-regulation of Egr1 was detected by immunoblotting in pSUPER.retro.neo+GFP tet/Egr1 clones ( 6A ) but not in mock transfected clones ( 6B ). The graph compares the signal intensities obtained from Egr1 bands.
    Figure Legend Snippet: To achieve an inducible knockdown of Egr1, we firstly transfected SK-HEP-1 cells with pCDNA6/TR which express tet repressor. Stable cells were selected using Blasticidin before co-transfecting SK-HEP-1 T-REx cells with tetracycline–regulated pSUPER.retro.neo+GFP tet RNAi construct, which was directed against a sequence containing Egr1. Stable cells were selected using G418. After selection, clones were analyzed for downregulation of Egr1 after 24 h of tetracycline treatment. Ten-fold down-regulation of Egr1 was detected by immunoblotting in pSUPER.retro.neo+GFP tet/Egr1 clones ( 6A ) but not in mock transfected clones ( 6B ). The graph compares the signal intensities obtained from Egr1 bands.

    Techniques Used: Transfection, Construct, Sequencing, Selection, Clone Assay, Western Blot

    SK-HEP-1 TREx cells were obtained by stable transfection of pCDNA-6/TR. Then SK-HEP-1 TREx cells were transfected with recombinant pSUPER-retro.neo+GFP vector expressing Egr-1 targeted shRNA or empty pSUPER-retro.neo+GFP vector using Fugene as described in . Knockdown of Egr1 in Egr1-shRNA-expressing SK-HEP-1 stable cell lines were induced by tetracycline ( 7A ). Equal amount of tetracycline induced and un-induced Egr1-shRNA-expressing SK-HEP-1 cells were used for migration and invasion assays in the presence and absence of HGF ( 7B, 7C ). Results are expressed as fold differences of matrigel invaded cells and are representative of two or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups. Zymographic gel showing active MMP-2 and-9 bands in 24 h conditioned medium of cultured Egr1-shRNA-expressing SK-HEP-1 cells left untreated or stimulated with tetracycline and/or HGF ( 7D, 7E ). Protein levels of MT1-MMP were studied in tetracyclin and/or HGF-induced SK-HEP-1-Egr1 cells ( 7F ). Densitometric analysis of gelatin zymography and immunoblots showed that HGF induces MT1-MMP expression and MMP-2 and -9 activation, whereas silencing of Egr1 blocks HGF-induced MMPs up-regulation.
    Figure Legend Snippet: SK-HEP-1 TREx cells were obtained by stable transfection of pCDNA-6/TR. Then SK-HEP-1 TREx cells were transfected with recombinant pSUPER-retro.neo+GFP vector expressing Egr-1 targeted shRNA or empty pSUPER-retro.neo+GFP vector using Fugene as described in . Knockdown of Egr1 in Egr1-shRNA-expressing SK-HEP-1 stable cell lines were induced by tetracycline ( 7A ). Equal amount of tetracycline induced and un-induced Egr1-shRNA-expressing SK-HEP-1 cells were used for migration and invasion assays in the presence and absence of HGF ( 7B, 7C ). Results are expressed as fold differences of matrigel invaded cells and are representative of two or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups. Zymographic gel showing active MMP-2 and-9 bands in 24 h conditioned medium of cultured Egr1-shRNA-expressing SK-HEP-1 cells left untreated or stimulated with tetracycline and/or HGF ( 7D, 7E ). Protein levels of MT1-MMP were studied in tetracyclin and/or HGF-induced SK-HEP-1-Egr1 cells ( 7F ). Densitometric analysis of gelatin zymography and immunoblots showed that HGF induces MT1-MMP expression and MMP-2 and -9 activation, whereas silencing of Egr1 blocks HGF-induced MMPs up-regulation.

    Techniques Used: Stable Transfection, Transfection, Recombinant, Plasmid Preparation, Expressing, shRNA, Migration, Cell Culture, Zymography, Western Blot, Activation Assay

    The effect of Egr1 on the cell invasion of HCC cell lines were examined using SNU-449 transiently transfected with pCMV-6-AC-GFP Egr1 overexpression vector ( 8A ). Equal amounts of wild type and Egr1 overexpressing SNU-449 cells were used for migration ( 8B ) and invasion ( 8C ) assay in the presence and absence of HGF. Results are expressed as fold differences of matrigel invaded cells. Zymographic gel showing the active MMP-9 bands in 24 h conditioned medium of cultured Egr1-overexpressing SNU-449 cells ( 8D ). The graph compares the signal intensities obtained from zymography gels.
    Figure Legend Snippet: The effect of Egr1 on the cell invasion of HCC cell lines were examined using SNU-449 transiently transfected with pCMV-6-AC-GFP Egr1 overexpression vector ( 8A ). Equal amounts of wild type and Egr1 overexpressing SNU-449 cells were used for migration ( 8B ) and invasion ( 8C ) assay in the presence and absence of HGF. Results are expressed as fold differences of matrigel invaded cells. Zymographic gel showing the active MMP-9 bands in 24 h conditioned medium of cultured Egr1-overexpressing SNU-449 cells ( 8D ). The graph compares the signal intensities obtained from zymography gels.

    Techniques Used: Transfection, Over Expression, Plasmid Preparation, Migration, Cell Culture, Zymography

    The effects of heparin and HGF on Egr1 expression in Egr1 over-expressing SNU-449 cells was analyzed by immunoblotting ( 9A ). Following serum deprivation, Egr1 overexpressed SN-449 cells were left untreated (0) or treated with HGF in the presence or absence of heparin, then invasion and migration assays were performed using the Roche xCELLigence System ( 9B, 9C ). Results are representative of three or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.
    Figure Legend Snippet: The effects of heparin and HGF on Egr1 expression in Egr1 over-expressing SNU-449 cells was analyzed by immunoblotting ( 9A ). Following serum deprivation, Egr1 overexpressed SN-449 cells were left untreated (0) or treated with HGF in the presence or absence of heparin, then invasion and migration assays were performed using the Roche xCELLigence System ( 9B, 9C ). Results are representative of three or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

    Techniques Used: Expressing, Western Blot, Migration

    Activation of HGF/c-Met signaling in HCC cells leads to MAPK signaling (i), and up-regulation of Egr1 (ii), which in turn induces MMP-2 and MMP-9 activation and MT1-MMP expression (iii), and increased cell motility and invasion (iv). Heparin treatment results in the inhibition of HGF-induced cellular invasion via repression of HGF-induced c-Met activation, as well as inhibition of MAPK signaling, downregulation of Egr1 expression, and MMP activation (v).
    Figure Legend Snippet: Activation of HGF/c-Met signaling in HCC cells leads to MAPK signaling (i), and up-regulation of Egr1 (ii), which in turn induces MMP-2 and MMP-9 activation and MT1-MMP expression (iii), and increased cell motility and invasion (iv). Heparin treatment results in the inhibition of HGF-induced cellular invasion via repression of HGF-induced c-Met activation, as well as inhibition of MAPK signaling, downregulation of Egr1 expression, and MMP activation (v).

    Techniques Used: Activation Assay, Expressing, Inhibition

    anti egr 1  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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  • 95

    Structured Review

    Cell Signaling Technology Inc anti egr 1
    Protocols of experimental grouping and reagent administering. (a) Protocol used to investigate role of ERK1/2 and <t>Egr-1</t> in extracellular-calcium-containing-H/R injury. (b) Protocol used to investigate role of PKC α /ERK1/2/Egr-1 in extracellular-calcium-containing-H/R injury. (c) Protocol used to investigate role of ERK1/2 and Egr-1 in extracellular-calcium-free-H/R injury. (d) Protocol used to investigate role of cAMP/PKA in extracellular-calcium-free-H/R injury. CaCon, calcium-containing normoxia; CaH/R, calcium-containing H/R; 0CaCon, calcium-free normoxic control; 0CaH/R, calcium-free H/R.
    Anti Egr 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti egr 1/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti egr 1 - by Bioz Stars, 2023-02
    95/100 stars

    Images

    1) Product Images from "N -n-Butyl Haloperidol Iodide Ameliorates Cardiomyocytes Hypoxia/Reoxygenation Injury by Extracellular Calcium-Dependent and -Independent Mechanisms"

    Article Title: N -n-Butyl Haloperidol Iodide Ameliorates Cardiomyocytes Hypoxia/Reoxygenation Injury by Extracellular Calcium-Dependent and -Independent Mechanisms

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2013/912310

    Protocols of experimental grouping and reagent administering. (a) Protocol used to investigate role of ERK1/2 and Egr-1 in extracellular-calcium-containing-H/R injury. (b) Protocol used to investigate role of PKC α /ERK1/2/Egr-1 in extracellular-calcium-containing-H/R injury. (c) Protocol used to investigate role of ERK1/2 and Egr-1 in extracellular-calcium-free-H/R injury. (d) Protocol used to investigate role of cAMP/PKA in extracellular-calcium-free-H/R injury. CaCon, calcium-containing normoxia; CaH/R, calcium-containing H/R; 0CaCon, calcium-free normoxic control; 0CaH/R, calcium-free H/R.
    Figure Legend Snippet: Protocols of experimental grouping and reagent administering. (a) Protocol used to investigate role of ERK1/2 and Egr-1 in extracellular-calcium-containing-H/R injury. (b) Protocol used to investigate role of PKC α /ERK1/2/Egr-1 in extracellular-calcium-containing-H/R injury. (c) Protocol used to investigate role of ERK1/2 and Egr-1 in extracellular-calcium-free-H/R injury. (d) Protocol used to investigate role of cAMP/PKA in extracellular-calcium-free-H/R injury. CaCon, calcium-containing normoxia; CaH/R, calcium-containing H/R; 0CaCon, calcium-free normoxic control; 0CaH/R, calcium-free H/R.

    Techniques Used:

    Effects of F 2 , Verapamil, and ERK1/2 inhibitors and activator on p-ERK1/2, total ERK1/2, and Egr-1 expression in extracellular-calcium-containing myocardial H/R by western-blot assay. (a) p-ERK1/2 and total ERK1/2; (b) Egr-1 protein. Quantitative densitometric data were expressed as percentages of the level observed in the CaCon group. All values are expressed as mean ± SEM of at least six individual experiments. * P < 0.05 versus CaCon group; # P < 0.05 versus CaH/R group; † P < 0.05 versus CaH/R+F 2 group.
    Figure Legend Snippet: Effects of F 2 , Verapamil, and ERK1/2 inhibitors and activator on p-ERK1/2, total ERK1/2, and Egr-1 expression in extracellular-calcium-containing myocardial H/R by western-blot assay. (a) p-ERK1/2 and total ERK1/2; (b) Egr-1 protein. Quantitative densitometric data were expressed as percentages of the level observed in the CaCon group. All values are expressed as mean ± SEM of at least six individual experiments. * P < 0.05 versus CaCon group; # P < 0.05 versus CaH/R group; † P < 0.05 versus CaH/R+F 2 group.

    Techniques Used: Expressing, Western Blot

    Effects of F 2 , Verapamil, and PKC α inhibitor and activator on p-PKC α , total PKC α , p-ERK1/2, total ERK1/2, and Egr-1 expression in extracellular-calcium-containing myocardial H/R by western-blot assay. (a) p-PKC α and total PKC α protein levels; (b) p-ERK1/2 and total ERK1/2 protein levels; (c) Egr-1 protein levels. All values are expressed as mean ± S.E.M. of at least six individual experiments. * P < 0.05 versus CaCon group; # P < 0.05 versus CaH/R group; † P < 0.05 versus CaH/R+F 2 group.
    Figure Legend Snippet: Effects of F 2 , Verapamil, and PKC α inhibitor and activator on p-PKC α , total PKC α , p-ERK1/2, total ERK1/2, and Egr-1 expression in extracellular-calcium-containing myocardial H/R by western-blot assay. (a) p-PKC α and total PKC α protein levels; (b) p-ERK1/2 and total ERK1/2 protein levels; (c) Egr-1 protein levels. All values are expressed as mean ± S.E.M. of at least six individual experiments. * P < 0.05 versus CaCon group; # P < 0.05 versus CaH/R group; † P < 0.05 versus CaH/R+F 2 group.

    Techniques Used: Expressing, Western Blot

    Effects of F 2 , ERK1/2 inhibitors, and activator on p-ERK1/2, total ERK1/2, and Egr-1 expression in extracellular-calcium-free myocardial H/R by western-blot assay. All values are expressed as mean ± S.E.M. of at least six individual experiments. * P < 0.05 versus 0CaCon group; # P < 0.05 versus 0CaH/R group; † P < 0.05 versus 0CaH/R+F 2 group.
    Figure Legend Snippet: Effects of F 2 , ERK1/2 inhibitors, and activator on p-ERK1/2, total ERK1/2, and Egr-1 expression in extracellular-calcium-free myocardial H/R by western-blot assay. All values are expressed as mean ± S.E.M. of at least six individual experiments. * P < 0.05 versus 0CaCon group; # P < 0.05 versus 0CaH/R group; † P < 0.05 versus 0CaH/R+F 2 group.

    Techniques Used: Expressing, Western Blot

    rabbit anti egr 1 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti egr 1 antibody
    (A) HMEC-1s and HUVECs were incubated with SW480-derived EVs (1 µg/mL) or untreated control. mRNA was isolated from untreated control cells or cells treated with EVs for 0, 0.5, 1, 2, and 4 h and analyzed using real time RT-PCR (n = 3). Values represent <t>Egr-1</t> mRNA/GAPDH mRNA normalized to untreated control cells. (B) HMEC-1s were treated with EVs (1 µg/mL) derived from HCT116 colorectal carcinoma, A549 lung adenocarcinoma, HT1080 fibrosarcoma, PC3 prostate carcinoma, SH-SY5Y neuroblastoma, and BEAS-2B normal bronchial epithelial cells for 0.5 h (n = 3). (C, D) In HMEC-1s, nuclear translocation of Egr-1 protein after stimulation with SW480-derived EVs (1 µg/mL) for 0.5, 1, 2, and 4 h was analyzed under confocal microscopy (n = 3). Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. Co-localized fluorescence signals (purple) indicate the translocation of Egr-1 into the nucleus. Representative photographs are shown in panel C. Scale bars represent 30 µm. The percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus colocalized signals over that of total cells (D). Data are represented as mean ± SD. * P <0.05; ** P <0.01; ** P <0.001; n.s., not significant.
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    Images

    1) Product Images from "Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways"

    Article Title: Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115170

    (A) HMEC-1s and HUVECs were incubated with SW480-derived EVs (1 µg/mL) or untreated control. mRNA was isolated from untreated control cells or cells treated with EVs for 0, 0.5, 1, 2, and 4 h and analyzed using real time RT-PCR (n = 3). Values represent Egr-1 mRNA/GAPDH mRNA normalized to untreated control cells. (B) HMEC-1s were treated with EVs (1 µg/mL) derived from HCT116 colorectal carcinoma, A549 lung adenocarcinoma, HT1080 fibrosarcoma, PC3 prostate carcinoma, SH-SY5Y neuroblastoma, and BEAS-2B normal bronchial epithelial cells for 0.5 h (n = 3). (C, D) In HMEC-1s, nuclear translocation of Egr-1 protein after stimulation with SW480-derived EVs (1 µg/mL) for 0.5, 1, 2, and 4 h was analyzed under confocal microscopy (n = 3). Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. Co-localized fluorescence signals (purple) indicate the translocation of Egr-1 into the nucleus. Representative photographs are shown in panel C. Scale bars represent 30 µm. The percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus colocalized signals over that of total cells (D). Data are represented as mean ± SD. * P <0.05; ** P <0.01; ** P <0.001; n.s., not significant.
    Figure Legend Snippet: (A) HMEC-1s and HUVECs were incubated with SW480-derived EVs (1 µg/mL) or untreated control. mRNA was isolated from untreated control cells or cells treated with EVs for 0, 0.5, 1, 2, and 4 h and analyzed using real time RT-PCR (n = 3). Values represent Egr-1 mRNA/GAPDH mRNA normalized to untreated control cells. (B) HMEC-1s were treated with EVs (1 µg/mL) derived from HCT116 colorectal carcinoma, A549 lung adenocarcinoma, HT1080 fibrosarcoma, PC3 prostate carcinoma, SH-SY5Y neuroblastoma, and BEAS-2B normal bronchial epithelial cells for 0.5 h (n = 3). (C, D) In HMEC-1s, nuclear translocation of Egr-1 protein after stimulation with SW480-derived EVs (1 µg/mL) for 0.5, 1, 2, and 4 h was analyzed under confocal microscopy (n = 3). Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. Co-localized fluorescence signals (purple) indicate the translocation of Egr-1 into the nucleus. Representative photographs are shown in panel C. Scale bars represent 30 µm. The percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus colocalized signals over that of total cells (D). Data are represented as mean ± SD. * P <0.05; ** P <0.01; ** P <0.001; n.s., not significant.

    Techniques Used: Incubation, Derivative Assay, Isolation, Quantitative RT-PCR, Translocation Assay, Confocal Microscopy, Staining, Fluorescence

    (A) HMEC-1s were transfected with 50 nM of scrambled siRNA or Egr-1 siRNA-1, Egr-1 siRNA-2, or Egr-1 siRNA-3. mRNAs were isolated from the cells after 48 h and the level of Egr-1 mRNA was analyzed using real time RT-PCR (n = 3). (B) Nuclear translocation of Egr-1 protein after stimulation with SW480-derived EVs (1 µg/mL) for 1 h was analyzed in scrambled siRNA (50 nM) or Egr-1 siRNA-1 (50 nM) transfected HMEC-1s (n = 3). (C, D) Confluent HMEC-1s transfected with scrambled siRNA (50 nM) or Egr-1 siRNA-1 (50 nM) were scratched and treated with SW480-derived EVs (1 µg/mL); then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). The number of migrated cells in the denuded zone of each group is shown in panel D. Scale bars represent 100 µm. Data are represented as mean ± SD. * P <0.05; ** P <0.01; *** P <0.001; n.s., not significant.
    Figure Legend Snippet: (A) HMEC-1s were transfected with 50 nM of scrambled siRNA or Egr-1 siRNA-1, Egr-1 siRNA-2, or Egr-1 siRNA-3. mRNAs were isolated from the cells after 48 h and the level of Egr-1 mRNA was analyzed using real time RT-PCR (n = 3). (B) Nuclear translocation of Egr-1 protein after stimulation with SW480-derived EVs (1 µg/mL) for 1 h was analyzed in scrambled siRNA (50 nM) or Egr-1 siRNA-1 (50 nM) transfected HMEC-1s (n = 3). (C, D) Confluent HMEC-1s transfected with scrambled siRNA (50 nM) or Egr-1 siRNA-1 (50 nM) were scratched and treated with SW480-derived EVs (1 µg/mL); then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). The number of migrated cells in the denuded zone of each group is shown in panel D. Scale bars represent 100 µm. Data are represented as mean ± SD. * P <0.05; ** P <0.01; *** P <0.001; n.s., not significant.

    Techniques Used: Transfection, Isolation, Quantitative RT-PCR, Translocation Assay, Derivative Assay

    (A, B) HMEC-1s were pretreated with signaling inhibitors for 1 h and then stimulated for 1 h with SW480-derived EVs (1 µg/mL). Nuclear translocation of Egr-1 protein was analyzed using confocal microscopy (n = 3). Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. Co-localized fluorescence signals (purple) indicate the translocation of Egr-1 into the nucleus. Representative photographs are shown in panel A. The percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus colocalized signals over total cells (B). (C, D) Confluent HMEC-1s were scratched and treated with SW480-derived EVs (1 µg/mL) in the presence or absence of signaling inhibitors; then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). Representative photographs of confocal microscopic imaging are shown in panel C and the number of migrated cells in the denuded zone of each group is shown in panel D. ERK1/2 inhibitor, PD98059 (20 µM); p38 MAPK inhibitor, SB203580 (10 µM); JNK inhibitor, SP600125 (20 µM); Akt inhibitor, BML-257 (20 µM). (E, F) C57BL/6 mice were subcutaneously injected with Matrigel containing SW480-derived EVs (20 µg) with PD98059 (20 µM) or SP600125 (20 µM). After 7 days, whole-mount staining of Matrigel with anti-CD31 antibody was conducted (n = 5). Representative confocal Z-stack photographs of whole mounts stained for CD31 (green) are shown in panel E. Fluorescence intensities of CD31 in the Z-stack plane of the Matrigel were measured as described in the (F). Scale bars in panels A, C, and E represent 30, 100, and 100 µm, respectively. Data are represented as mean ± SD. *** P <0.001.
    Figure Legend Snippet: (A, B) HMEC-1s were pretreated with signaling inhibitors for 1 h and then stimulated for 1 h with SW480-derived EVs (1 µg/mL). Nuclear translocation of Egr-1 protein was analyzed using confocal microscopy (n = 3). Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. Co-localized fluorescence signals (purple) indicate the translocation of Egr-1 into the nucleus. Representative photographs are shown in panel A. The percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus colocalized signals over total cells (B). (C, D) Confluent HMEC-1s were scratched and treated with SW480-derived EVs (1 µg/mL) in the presence or absence of signaling inhibitors; then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). Representative photographs of confocal microscopic imaging are shown in panel C and the number of migrated cells in the denuded zone of each group is shown in panel D. ERK1/2 inhibitor, PD98059 (20 µM); p38 MAPK inhibitor, SB203580 (10 µM); JNK inhibitor, SP600125 (20 µM); Akt inhibitor, BML-257 (20 µM). (E, F) C57BL/6 mice were subcutaneously injected with Matrigel containing SW480-derived EVs (20 µg) with PD98059 (20 µM) or SP600125 (20 µM). After 7 days, whole-mount staining of Matrigel with anti-CD31 antibody was conducted (n = 5). Representative confocal Z-stack photographs of whole mounts stained for CD31 (green) are shown in panel E. Fluorescence intensities of CD31 in the Z-stack plane of the Matrigel were measured as described in the (F). Scale bars in panels A, C, and E represent 30, 100, and 100 µm, respectively. Data are represented as mean ± SD. *** P <0.001.

    Techniques Used: Derivative Assay, Translocation Assay, Confocal Microscopy, Staining, Fluorescence, Imaging, Injection

    (A) HMEC-1s were treated with DiI-labeled SW480-derived EVs (1 µg/mL) for 1 h in the presence or absence of MβCD (10 mM) (n = 3). SW480-derived EVs and nuclei were stained with DiI (red) and Hoechst (blue) respectively. Representative photographs are shown in panel A. Scale bars represent 10 µm. (B) Confluent HMEC-1s were scratched and treated with SW480-derived EVs (1 µg/mL) in the presence or absence of MβCD (10 mM); then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). (C, D) HMEC-1s were pretreated with MβCD (10 mM) for 1 h and then stimulated with SW480-derived EVs (1 µg/mL) for 1 h. Scale bars represent 30 µm. Nuclear translocation of Egr-1 protein was analyzed using confocal microscopy and the percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus co-localized signals over that of total cells (n = 3). Data are represented as mean ± SD. *** P <0.001.
    Figure Legend Snippet: (A) HMEC-1s were treated with DiI-labeled SW480-derived EVs (1 µg/mL) for 1 h in the presence or absence of MβCD (10 mM) (n = 3). SW480-derived EVs and nuclei were stained with DiI (red) and Hoechst (blue) respectively. Representative photographs are shown in panel A. Scale bars represent 10 µm. (B) Confluent HMEC-1s were scratched and treated with SW480-derived EVs (1 µg/mL) in the presence or absence of MβCD (10 mM); then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). (C, D) HMEC-1s were pretreated with MβCD (10 mM) for 1 h and then stimulated with SW480-derived EVs (1 µg/mL) for 1 h. Scale bars represent 30 µm. Nuclear translocation of Egr-1 protein was analyzed using confocal microscopy and the percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus co-localized signals over that of total cells (n = 3). Data are represented as mean ± SD. *** P <0.001.

    Techniques Used: Labeling, Derivative Assay, Staining, Translocation Assay, Confocal Microscopy

    egr1 primary antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr1 primary antibody
    A. WT1 mRNA (+KTS and –KTS) splice variant PCR products expressed in LβT2 cells. Whole cell RNA was extracted from LβT2 cells, reverse transcribed to cDNA and quantified by real-time PCR, using specific primers to detect +KTS and –KTS splice variants, then displayed on a 1% agarose gel. Bands corresponding to the products for +KTS (301bp) and –KTS (292 bp) WT1 were detected in 4 independent samples of mRNA. B. WT1 and <t>Egr1</t> protein expression in LβT2 cells. Cell proteins (30μg) were separated on 10% polyacrylamide-SDS gels, then analyzed by immunoblotting with specific antibodies for WT1, Egr1 and β-actin. Specific proteins were detected in the same samples of untreated LβT2 cells. C. Chromatin association of WT1 with the endogenous LHβ promoter in LβT2 cells. The association of WT1 and RNA Polymerase II with the LHβ promoter in untreated LβT2 cells was measured by Chromatin immunoprecipitation assays with antibodies against WT1 and phosphorylated RNApol II, as well as control (no Antibody). LHβ promoter occupancy was measured by quantitative real time PCR using primers specific for the LHβ promoter, and normalized for chromatin input in each sample. In this study, background binding (no Antibody) was set at 100% and association of RNA Polymerase II and WT1 are expressed relative to background values. Association was measured in 3 independent experiments with duplicate samples.
    Egr1 Primary Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A New Role for Wilms Tumor Protein 1: Differential Activities of + KTS and –KTS Variants to Regulate LHβ Transcription"

    Article Title: A New Role for Wilms Tumor Protein 1: Differential Activities of + KTS and –KTS Variants to Regulate LHβ Transcription

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116825

    A. WT1 mRNA (+KTS and –KTS) splice variant PCR products expressed in LβT2 cells. Whole cell RNA was extracted from LβT2 cells, reverse transcribed to cDNA and quantified by real-time PCR, using specific primers to detect +KTS and –KTS splice variants, then displayed on a 1% agarose gel. Bands corresponding to the products for +KTS (301bp) and –KTS (292 bp) WT1 were detected in 4 independent samples of mRNA. B. WT1 and Egr1 protein expression in LβT2 cells. Cell proteins (30μg) were separated on 10% polyacrylamide-SDS gels, then analyzed by immunoblotting with specific antibodies for WT1, Egr1 and β-actin. Specific proteins were detected in the same samples of untreated LβT2 cells. C. Chromatin association of WT1 with the endogenous LHβ promoter in LβT2 cells. The association of WT1 and RNA Polymerase II with the LHβ promoter in untreated LβT2 cells was measured by Chromatin immunoprecipitation assays with antibodies against WT1 and phosphorylated RNApol II, as well as control (no Antibody). LHβ promoter occupancy was measured by quantitative real time PCR using primers specific for the LHβ promoter, and normalized for chromatin input in each sample. In this study, background binding (no Antibody) was set at 100% and association of RNA Polymerase II and WT1 are expressed relative to background values. Association was measured in 3 independent experiments with duplicate samples.
    Figure Legend Snippet: A. WT1 mRNA (+KTS and –KTS) splice variant PCR products expressed in LβT2 cells. Whole cell RNA was extracted from LβT2 cells, reverse transcribed to cDNA and quantified by real-time PCR, using specific primers to detect +KTS and –KTS splice variants, then displayed on a 1% agarose gel. Bands corresponding to the products for +KTS (301bp) and –KTS (292 bp) WT1 were detected in 4 independent samples of mRNA. B. WT1 and Egr1 protein expression in LβT2 cells. Cell proteins (30μg) were separated on 10% polyacrylamide-SDS gels, then analyzed by immunoblotting with specific antibodies for WT1, Egr1 and β-actin. Specific proteins were detected in the same samples of untreated LβT2 cells. C. Chromatin association of WT1 with the endogenous LHβ promoter in LβT2 cells. The association of WT1 and RNA Polymerase II with the LHβ promoter in untreated LβT2 cells was measured by Chromatin immunoprecipitation assays with antibodies against WT1 and phosphorylated RNApol II, as well as control (no Antibody). LHβ promoter occupancy was measured by quantitative real time PCR using primers specific for the LHβ promoter, and normalized for chromatin input in each sample. In this study, background binding (no Antibody) was set at 100% and association of RNA Polymerase II and WT1 are expressed relative to background values. Association was measured in 3 independent experiments with duplicate samples.

    Techniques Used: Variant Assay, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Western Blot, Chromatin Immunoprecipitation, Binding Assay

    A. WT1 (-KTS) and (+ KTS) mRNA variant levels in LβT2 cells treated with 50nM GnRH for 90min. RNA was extracted and mRNAs levels measured by RT-PCR and normalized against GAPDH mRNA. Values for WT1 mRNAs are shown for both Reverse Transcriptase (RT) and no RT (negative control) conditions. Note that for no RT, the Y axis is interrupted and expanded to show the low values. Data is the mean ± SEM from 5–7 experiments. * = P<0.05, -GnRH vs +GnRH; ** = P<0.001, -GnRH vs +GnRH B. WT1 and Egr1 protein levels in LβT2 cells treated with 50nM GnRH for 0 to 3.5 h. The experiment was performed three times. Upper panel: Cells were lysed after GnRH treatment and proteins (30μg) were separated by 10% SDS-PAGE, then detected with antibodies against WT1 or Egr1. Immunoblotting for β-actin was performed on the same blots as WT1 and Egr1, and used for normalization of these proteins quantified by densitometry analysis. A representative blot is shown. Lower panel: Quantification of protein bands was performed with densitometry, and normalized protein levels are shown from combined experiments. Bands for Egr1 proteins were not detected (ND) in any blot at time zero without GnRH.
    Figure Legend Snippet: A. WT1 (-KTS) and (+ KTS) mRNA variant levels in LβT2 cells treated with 50nM GnRH for 90min. RNA was extracted and mRNAs levels measured by RT-PCR and normalized against GAPDH mRNA. Values for WT1 mRNAs are shown for both Reverse Transcriptase (RT) and no RT (negative control) conditions. Note that for no RT, the Y axis is interrupted and expanded to show the low values. Data is the mean ± SEM from 5–7 experiments. * = P<0.05, -GnRH vs +GnRH; ** = P<0.001, -GnRH vs +GnRH B. WT1 and Egr1 protein levels in LβT2 cells treated with 50nM GnRH for 0 to 3.5 h. The experiment was performed three times. Upper panel: Cells were lysed after GnRH treatment and proteins (30μg) were separated by 10% SDS-PAGE, then detected with antibodies against WT1 or Egr1. Immunoblotting for β-actin was performed on the same blots as WT1 and Egr1, and used for normalization of these proteins quantified by densitometry analysis. A representative blot is shown. Lower panel: Quantification of protein bands was performed with densitometry, and normalized protein levels are shown from combined experiments. Bands for Egr1 proteins were not detected (ND) in any blot at time zero without GnRH.

    Techniques Used: Variant Assay, Reverse Transcription Polymerase Chain Reaction, Negative Control, SDS Page, Western Blot

    LβT2 cells were incubated with or without 50 nM GnRH and collected every 10 min for 120 min. ChIP assays were performed using antibody against WT1, Egr1 and phosphorylated pRNA Pol II. LHβ promoter occupancy was measured by quantitative real time PCR using primers for the LHβ promoter. The experiment was performed three times with duplicate samples and replicate PCR measurements in each sample. Data are presented as the mean + SEM and expressed as LHβ promoter occupancy relative to basal (no GnRH at time zero) binding.
    Figure Legend Snippet: LβT2 cells were incubated with or without 50 nM GnRH and collected every 10 min for 120 min. ChIP assays were performed using antibody against WT1, Egr1 and phosphorylated pRNA Pol II. LHβ promoter occupancy was measured by quantitative real time PCR using primers for the LHβ promoter. The experiment was performed three times with duplicate samples and replicate PCR measurements in each sample. Data are presented as the mean + SEM and expressed as LHβ promoter occupancy relative to basal (no GnRH at time zero) binding.

    Techniques Used: Incubation, Real-time Polymerase Chain Reaction, Binding Assay

    LβT2 cells were transfected with siRNA against WT1 and a non-targeting siRNA as a control. After 72 h of incubation, cells were treated with or without GnRH for 1.5 h, followed by cell lysate collection, RNA extraction and western blot analysis (30 μg protein) on 10% PAGE-SDS gels. WT1 protein was detected by immunoblotting with specific antibodies for WT1, and normalized for β-actin on the same blot. A. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where the WT1 (-KTS) variant mRNA was reduced. B. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where both the WT1 (-KTS) and WT1 (+KTS) variant mRNAs were reduced. For each condition, the experiment was performed twice with 5 replicates. LHβ and Egr1 primary transcript mRNAs were normalized for GAPDH mRNA in the same sample. Control samples contained non-targeting siRNA. * p<.05 -GnRH vs +GnRH, in either Control siRNA or WT1 siRNA treatments. Values are mean ± SEM for 5 replicates. # = p<.05 -GnRH Control siRNA vs –GnRH WT1 siRNA, or p<.05+GnRH Control siRNA vs +GnRH WT1 siRNA.
    Figure Legend Snippet: LβT2 cells were transfected with siRNA against WT1 and a non-targeting siRNA as a control. After 72 h of incubation, cells were treated with or without GnRH for 1.5 h, followed by cell lysate collection, RNA extraction and western blot analysis (30 μg protein) on 10% PAGE-SDS gels. WT1 protein was detected by immunoblotting with specific antibodies for WT1, and normalized for β-actin on the same blot. A. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where the WT1 (-KTS) variant mRNA was reduced. B. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where both the WT1 (-KTS) and WT1 (+KTS) variant mRNAs were reduced. For each condition, the experiment was performed twice with 5 replicates. LHβ and Egr1 primary transcript mRNAs were normalized for GAPDH mRNA in the same sample. Control samples contained non-targeting siRNA. * p<.05 -GnRH vs +GnRH, in either Control siRNA or WT1 siRNA treatments. Values are mean ± SEM for 5 replicates. # = p<.05 -GnRH Control siRNA vs –GnRH WT1 siRNA, or p<.05+GnRH Control siRNA vs +GnRH WT1 siRNA.

    Techniques Used: Transfection, Incubation, RNA Extraction, Western Blot, Expressing, Variant Assay

    WT1 (-KTS) may associate directly with the 3’Egr1 site of the LHβ promoter to stimulate basal promoter activity, but in the presence of GnRH stimulates Egr1 expression. Egr1 then binds to both the 3’- and 5’-Egr1 sites on the promoter and further increases LHβ transcription. WT1 (+KTS) may associate with the 3’-Egr1 site, but also requires the SF1 site for biological activity. Decreased expression of WT1 (+KTS) would stimulate basal LHβ expression as the suppressor is reduced. The isoforms likely compete for association to the LHβ promoter at the same sites.
    Figure Legend Snippet: WT1 (-KTS) may associate directly with the 3’Egr1 site of the LHβ promoter to stimulate basal promoter activity, but in the presence of GnRH stimulates Egr1 expression. Egr1 then binds to both the 3’- and 5’-Egr1 sites on the promoter and further increases LHβ transcription. WT1 (+KTS) may associate with the 3’-Egr1 site, but also requires the SF1 site for biological activity. Decreased expression of WT1 (+KTS) would stimulate basal LHβ expression as the suppressor is reduced. The isoforms likely compete for association to the LHβ promoter at the same sites.

    Techniques Used: Activity Assay, Expressing

    rabbit monoclonal antibodies egr  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal antibodies egr
    Growth-quiescent SMCs were treated with MMP inhibitor GM6001+ (25 µM) and TAPI-1 (10 µM) for 30 min before stimulation with IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). ( A ) <t>Egr-1</t> mRNA levels in GM6001+ or GM6001- treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( B ) Western blotting for Egr-1 or beta-actin in total extracts of SMCs treated with GM6001+ or GM6001-. ( C ) Egr-1 mRNA levels in TAPI-1 treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( D ) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with TAPI-1. DMSO was used as a carrier. ( E ) Western blotting for ADAM17, ADAM10, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ADAM17 siRNA (0.1 µM) and treated with IL-1beta for 30 min.
    Rabbit Monoclonal Antibodies Egr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "IL-1beta Signals through the EGF Receptor and Activates Egr-1 through MMP-ADAM"

    Article Title: IL-1beta Signals through the EGF Receptor and Activates Egr-1 through MMP-ADAM

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039811

    Growth-quiescent SMCs were treated with MMP inhibitor GM6001+ (25 µM) and TAPI-1 (10 µM) for 30 min before stimulation with IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). ( A ) Egr-1 mRNA levels in GM6001+ or GM6001- treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( B ) Western blotting for Egr-1 or beta-actin in total extracts of SMCs treated with GM6001+ or GM6001-. ( C ) Egr-1 mRNA levels in TAPI-1 treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( D ) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with TAPI-1. DMSO was used as a carrier. ( E ) Western blotting for ADAM17, ADAM10, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ADAM17 siRNA (0.1 µM) and treated with IL-1beta for 30 min.
    Figure Legend Snippet: Growth-quiescent SMCs were treated with MMP inhibitor GM6001+ (25 µM) and TAPI-1 (10 µM) for 30 min before stimulation with IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). ( A ) Egr-1 mRNA levels in GM6001+ or GM6001- treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( B ) Western blotting for Egr-1 or beta-actin in total extracts of SMCs treated with GM6001+ or GM6001-. ( C ) Egr-1 mRNA levels in TAPI-1 treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( D ) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with TAPI-1. DMSO was used as a carrier. ( E ) Western blotting for ADAM17, ADAM10, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ADAM17 siRNA (0.1 µM) and treated with IL-1beta for 30 min.

    Techniques Used: Western Blot, Transfection

    Growth-quiescent SMCs were incubated with EGFR inhibitors AG1478 (5 µM) and PD153035 (5 µM) for 30 min, and exposed to IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). ( A ) Egr-1 mRNA levels in AG1478- and PD153035-treated SMCs incubated with IL-1beta by qPCR. Data were normalized to beta-actin. ( B ) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with AG1478 or PD153035. ( C ) Western blotting for EGFR, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with EGFR siRNA (0.1 µM) and treated with IL-1beta for 30 min. ( D ) Western blotting for ErbB4, EGFR, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ErbB4 siRNA (0.1 µM) and treated with IL-1beta for 30 min. ( E ) Egr-1 mRNA levels in AG825 (10 µM for 30 min)-treated SMCs incubated with IL-1beta for 30 min by qPCR. Data were normalized to beta-actin. ( F ) Western blotting for Egr-1 or beta-actin in total extracts of SMCs pretreated with AG825 (10 µM for 30 min) then stimulated with IL-1beta for another 30 min.
    Figure Legend Snippet: Growth-quiescent SMCs were incubated with EGFR inhibitors AG1478 (5 µM) and PD153035 (5 µM) for 30 min, and exposed to IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). ( A ) Egr-1 mRNA levels in AG1478- and PD153035-treated SMCs incubated with IL-1beta by qPCR. Data were normalized to beta-actin. ( B ) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with AG1478 or PD153035. ( C ) Western blotting for EGFR, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with EGFR siRNA (0.1 µM) and treated with IL-1beta for 30 min. ( D ) Western blotting for ErbB4, EGFR, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ErbB4 siRNA (0.1 µM) and treated with IL-1beta for 30 min. ( E ) Egr-1 mRNA levels in AG825 (10 µM for 30 min)-treated SMCs incubated with IL-1beta for 30 min by qPCR. Data were normalized to beta-actin. ( F ) Western blotting for Egr-1 or beta-actin in total extracts of SMCs pretreated with AG825 (10 µM for 30 min) then stimulated with IL-1beta for another 30 min.

    Techniques Used: Incubation, Western Blot, Transfection

    ( A ) Growth-quiescent SMCs were stimulated with IL-1beta (10 ng/ml) for the indicated times. Total protein extracts were resolved by SDS-PAGE and subjected to Western blot analysis using antibodies for Egr-1, EGFR phospho-Tyr 845 , total EGFR and beta-actin. Alternatively, Western blotting for EGFR phospho-Tyr 845 or EGFR of total extracts of SMCs treated with ( B ) AG1478 (5 µM), ( C ) GM6001+ or GM6001- (25 µM) ( D ) TAPI-1 (10 µM), PD153035 (5 µM) for 30 min followed by IL-1beta stimulation for 5 min.
    Figure Legend Snippet: ( A ) Growth-quiescent SMCs were stimulated with IL-1beta (10 ng/ml) for the indicated times. Total protein extracts were resolved by SDS-PAGE and subjected to Western blot analysis using antibodies for Egr-1, EGFR phospho-Tyr 845 , total EGFR and beta-actin. Alternatively, Western blotting for EGFR phospho-Tyr 845 or EGFR of total extracts of SMCs treated with ( B ) AG1478 (5 µM), ( C ) GM6001+ or GM6001- (25 µM) ( D ) TAPI-1 (10 µM), PD153035 (5 µM) for 30 min followed by IL-1beta stimulation for 5 min.

    Techniques Used: SDS Page, Western Blot

    ( A ) Western blotting for Egr-1, ADAM17, EGFR, IL-1RI or beta-actin using total extracts of growth-quiescent wild-type or ADAM17-deficient mEFs treated with IL-1beta for 30 or 60 min. ( B ) Interaction of 125 I-IL-1beta with ADAM17WT and ADAM17-deficient cells. Growth-quiescent ADAM17WT and ADAM17−/− mEFs (1.8×10 4 cells/well) were incubated with increasing amounts of 125 I-IL-1beta in 1% BSA/PBS for 1 h at 4°C. The cells were washed and lysed with 1M NaOH prior to assessment of counts in an automated gamma-counter.
    Figure Legend Snippet: ( A ) Western blotting for Egr-1, ADAM17, EGFR, IL-1RI or beta-actin using total extracts of growth-quiescent wild-type or ADAM17-deficient mEFs treated with IL-1beta for 30 or 60 min. ( B ) Interaction of 125 I-IL-1beta with ADAM17WT and ADAM17-deficient cells. Growth-quiescent ADAM17WT and ADAM17−/− mEFs (1.8×10 4 cells/well) were incubated with increasing amounts of 125 I-IL-1beta in 1% BSA/PBS for 1 h at 4°C. The cells were washed and lysed with 1M NaOH prior to assessment of counts in an automated gamma-counter.

    Techniques Used: Western Blot, Incubation

    rabbit monoclonal anti egr1 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal anti egr1 antibody
    Oligonucleotides and plasmids.
    Rabbit Monoclonal Anti Egr1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Transcriptional Regulation of Flotillins by the Extracellularly Regulated Kinases and Retinoid X Receptor Complexes"

    Article Title: Transcriptional Regulation of Flotillins by the Extracellularly Regulated Kinases and Retinoid X Receptor Complexes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045514

    Oligonucleotides and plasmids.
    Figure Legend Snippet: Oligonucleotides and plasmids.

    Techniques Used: Luciferase, Plasmid Preparation, Expressing, Dominant Negative Mutation

    Flotillin promoter constructs F1-1330 (A, C, E) or F2-2130 (B, D, F) were cotransfected into Hela cells together with expression plasmids for Egr1, SRF, empty PSV control plasmid or with the ERK2 expression constructs, and the cells were lysed 48 h post-transfection. Relative luciferase activity of the control sample was set as 1. Values are mean ± standard deviation of at least 4 experiments measured in duplicates. ***p<0.001; **p<0.01; *p<0.05 vs. control.
    Figure Legend Snippet: Flotillin promoter constructs F1-1330 (A, C, E) or F2-2130 (B, D, F) were cotransfected into Hela cells together with expression plasmids for Egr1, SRF, empty PSV control plasmid or with the ERK2 expression constructs, and the cells were lysed 48 h post-transfection. Relative luciferase activity of the control sample was set as 1. Values are mean ± standard deviation of at least 4 experiments measured in duplicates. ***p<0.001; **p<0.01; *p<0.05 vs. control.

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

    Hela cells were transiently transfected with expression constructs for Egr1 or SRF. Empty PSV vector served as control. (A) Cell lysates were analyzed for flotillin-1 and -2 by Western blotting (left) and quantified (right). (B) RNA was isolated, transcribed into cDNA and analyzed by qPCR. Values are means ± standard deviation of 7 (Western Blot) or 4 (qPCR) experiments.*p<0.05, **, p<0.01 vs. control.
    Figure Legend Snippet: Hela cells were transiently transfected with expression constructs for Egr1 or SRF. Empty PSV vector served as control. (A) Cell lysates were analyzed for flotillin-1 and -2 by Western blotting (left) and quantified (right). (B) RNA was isolated, transcribed into cDNA and analyzed by qPCR. Values are means ± standard deviation of 7 (Western Blot) or 4 (qPCR) experiments.*p<0.05, **, p<0.01 vs. control.

    Techniques Used: Transfection, Expressing, Construct, Plasmid Preparation, Western Blot, Isolation, Standard Deviation

    Chromatin immunoprecipitations were carried out in Hela cells as described in . DNA/protein complexes were precipitated with antibodies against RXR, RAR, Egr1 or HRS (IgG control). Coprecipitated DNA fragments were amplified with primers specific for approximately 500 bp (flotillin-1) (A) or 400 bp (flotillin-2) (B) of genomic DNA directly upstream of the ATG start codon. Results are representative of two independent experiments.
    Figure Legend Snippet: Chromatin immunoprecipitations were carried out in Hela cells as described in . DNA/protein complexes were precipitated with antibodies against RXR, RAR, Egr1 or HRS (IgG control). Coprecipitated DNA fragments were amplified with primers specific for approximately 500 bp (flotillin-1) (A) or 400 bp (flotillin-2) (B) of genomic DNA directly upstream of the ATG start codon. Results are representative of two independent experiments.

    Techniques Used: Amplification

    A: mouse flotillin-2 proximal promoter sequence. The three putative binding sites for the RXR family and the two sites for Egr1 are shown in bold and underlined. The sequence shown in italics was deleted to remove the Egr1 binding sites. Mouse flotillin-2 promoter activity after PMA stimulation (B) or retinoic acid stimulation (C). Transfection and stimulation were performed as in .
    Figure Legend Snippet: A: mouse flotillin-2 proximal promoter sequence. The three putative binding sites for the RXR family and the two sites for Egr1 are shown in bold and underlined. The sequence shown in italics was deleted to remove the Egr1 binding sites. Mouse flotillin-2 promoter activity after PMA stimulation (B) or retinoic acid stimulation (C). Transfection and stimulation were performed as in .

    Techniques Used: Sequencing, Binding Assay, Activity Assay, Transfection

    egr1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr1
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    egr 1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr 1
    ( A ) IVT-synthesized <t>Egr-1</t> binds to ORF50 P probes. EMSA studies were performed using IVT-synthesized Egr-1 products and DIG-labeled ORF50 P probes (see ). ( B ) Mutations in the putative Egr-1 binding domain inhibit Egr-1 binding. EMSA experiments were performed using wt ORF50 P3 and ORF50 P8 probes as well as corresponding probes displaying mutations in the suspected Egr-1 binding domain ( ORF50 P3m and ORF50 P8m). ( C ) Nuclear lysates from KSHV-infected cells formed a complex with ORF50 P probes. BCBL-1 cells were synchronized in S phase of cell cycle according to earlier protocols , treated with 20 ng/ml TPA for 8 h, and lysed. Nuclear extracts containing Egr-1 proteins were used to perform EMSA studies using ORF50 P3 and ORF50 P8 probes. Specific Egr-1 binding was confirmed by performing supershifts using specific antibodies to Egr-1 ( lanes 4 and 8 ) or nonspecific IgGs ( lanes 3 and 7 ). The arrowhead indicates protein/DNA complex formation. Specific antibody/protein/DNA supershifts are denoted by the asterisk.
    Egr 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Resveratrol Inhibits KSHV Reactivation by Lowering the Levels of Cellular EGR-1"

    Article Title: Resveratrol Inhibits KSHV Reactivation by Lowering the Levels of Cellular EGR-1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033364

    ( A ) IVT-synthesized Egr-1 binds to ORF50 P probes. EMSA studies were performed using IVT-synthesized Egr-1 products and DIG-labeled ORF50 P probes (see ). ( B ) Mutations in the putative Egr-1 binding domain inhibit Egr-1 binding. EMSA experiments were performed using wt ORF50 P3 and ORF50 P8 probes as well as corresponding probes displaying mutations in the suspected Egr-1 binding domain ( ORF50 P3m and ORF50 P8m). ( C ) Nuclear lysates from KSHV-infected cells formed a complex with ORF50 P probes. BCBL-1 cells were synchronized in S phase of cell cycle according to earlier protocols , treated with 20 ng/ml TPA for 8 h, and lysed. Nuclear extracts containing Egr-1 proteins were used to perform EMSA studies using ORF50 P3 and ORF50 P8 probes. Specific Egr-1 binding was confirmed by performing supershifts using specific antibodies to Egr-1 ( lanes 4 and 8 ) or nonspecific IgGs ( lanes 3 and 7 ). The arrowhead indicates protein/DNA complex formation. Specific antibody/protein/DNA supershifts are denoted by the asterisk.
    Figure Legend Snippet: ( A ) IVT-synthesized Egr-1 binds to ORF50 P probes. EMSA studies were performed using IVT-synthesized Egr-1 products and DIG-labeled ORF50 P probes (see ). ( B ) Mutations in the putative Egr-1 binding domain inhibit Egr-1 binding. EMSA experiments were performed using wt ORF50 P3 and ORF50 P8 probes as well as corresponding probes displaying mutations in the suspected Egr-1 binding domain ( ORF50 P3m and ORF50 P8m). ( C ) Nuclear lysates from KSHV-infected cells formed a complex with ORF50 P probes. BCBL-1 cells were synchronized in S phase of cell cycle according to earlier protocols , treated with 20 ng/ml TPA for 8 h, and lysed. Nuclear extracts containing Egr-1 proteins were used to perform EMSA studies using ORF50 P3 and ORF50 P8 probes. Specific Egr-1 binding was confirmed by performing supershifts using specific antibodies to Egr-1 ( lanes 4 and 8 ) or nonspecific IgGs ( lanes 3 and 7 ). The arrowhead indicates protein/DNA complex formation. Specific antibody/protein/DNA supershifts are denoted by the asterisk.

    Techniques Used: Synthesized, Labeling, Binding Assay, Infection

    ( A ) BCBL-1 cells were synchronized in S phase and treated with 20 ng/ml of TPA for 8 h. ChIP assays were performed using 2 µg of specific antibodies to Egr-1 or nonspecific IgGs. Primers specifically targeting ORF50 P3 or ORF50 P8 (see ) were used to perform semi-quantitative PCR experiments on 1% of total DNA (input) and IP samples. Respective cDNA at 10, 15, 20, 25, and 30 cycles were removed and resolved on a 2% agarose gel. IP of BCBL-1 DNA using specific antibodies to histone H3 was used as positive controls. ( B ) A schematic representation of ORF50P used to make the deletions of the luciferase reporter constructs. The nucleotide locations correspond to the old KSHV genome sequence NC_003409 which has since been updated to NC_009333.1. Asterisks refer to the ORF50P3 and ORF50P8 locations, respectively. ( C ) Overexpression of Egr-1 activates ORF50P via interacting with ORF50P3 and ORF50P8 domains. HEK293 cells were co-transfected with a combination of three vectors, one from the following groups: (i) pcDNA3.1(+) or egr-1 /pcDNA3.1(+), (ii) the control vector, pRL-TK, and (iii) empty pGL3 vectors or pGL3 vectors encoding FL ORF50P or one of several deletions (Δ-2922 to -2044; Δ-2922 to -1322; Δ-2922 to -894; and Δ-2922 to -169). After 48 h post-transfection, the cells were lysed, and relative luciferase activity was monitored. Firefly luciferase was normalized to the corresponding Renilla luciferase activity. The luciferase activation of pGL3 by egr-1 /pcDNA3.1(+) was represented as 1-fold. Each point denotes the average ± SD of three experiments. Columns with different alphabets are statistically significant (P<0.05) by least significant difference (LSD).
    Figure Legend Snippet: ( A ) BCBL-1 cells were synchronized in S phase and treated with 20 ng/ml of TPA for 8 h. ChIP assays were performed using 2 µg of specific antibodies to Egr-1 or nonspecific IgGs. Primers specifically targeting ORF50 P3 or ORF50 P8 (see ) were used to perform semi-quantitative PCR experiments on 1% of total DNA (input) and IP samples. Respective cDNA at 10, 15, 20, 25, and 30 cycles were removed and resolved on a 2% agarose gel. IP of BCBL-1 DNA using specific antibodies to histone H3 was used as positive controls. ( B ) A schematic representation of ORF50P used to make the deletions of the luciferase reporter constructs. The nucleotide locations correspond to the old KSHV genome sequence NC_003409 which has since been updated to NC_009333.1. Asterisks refer to the ORF50P3 and ORF50P8 locations, respectively. ( C ) Overexpression of Egr-1 activates ORF50P via interacting with ORF50P3 and ORF50P8 domains. HEK293 cells were co-transfected with a combination of three vectors, one from the following groups: (i) pcDNA3.1(+) or egr-1 /pcDNA3.1(+), (ii) the control vector, pRL-TK, and (iii) empty pGL3 vectors or pGL3 vectors encoding FL ORF50P or one of several deletions (Δ-2922 to -2044; Δ-2922 to -1322; Δ-2922 to -894; and Δ-2922 to -169). After 48 h post-transfection, the cells were lysed, and relative luciferase activity was monitored. Firefly luciferase was normalized to the corresponding Renilla luciferase activity. The luciferase activation of pGL3 by egr-1 /pcDNA3.1(+) was represented as 1-fold. Each point denotes the average ± SD of three experiments. Columns with different alphabets are statistically significant (P<0.05) by least significant difference (LSD).

    Techniques Used: Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Luciferase, Construct, Sequencing, Over Expression, Transfection, Plasmid Preparation, Activity Assay, Activation Assay

    HEK293 cells were infected with KSHV at 5 MOI, incubated at 37°C at different time points up to 48 hPI, and lysed. ( A ) Expression of KSHV RTA and Egr-1 were elevated up to 6 hPI. SDS-PAGE was performed using uninfected ( lane 1 ) or KSHV-infected cell lysates ( lanes 2–7 ). The samples were run on a 10% gel and transferred to a PVDF membrane. Western blotting was performed using specific Egr-1 or KSHV RTA Abs. Abs targeting β-actin were used as internal controls. ( B ) Analysis of ORF50 and egr-1 transcription. KSHV-infected HEK293 cells were lysed and RNA was extracted. Next, cDNA was synthesized and subjected to quantitative real-time PCR analysis. Baseline expression of genes at 1 hPI was considered as 1-fold for comparisons. Each point denotes the average±S.D. ( error bars ) of three experiments.
    Figure Legend Snippet: HEK293 cells were infected with KSHV at 5 MOI, incubated at 37°C at different time points up to 48 hPI, and lysed. ( A ) Expression of KSHV RTA and Egr-1 were elevated up to 6 hPI. SDS-PAGE was performed using uninfected ( lane 1 ) or KSHV-infected cell lysates ( lanes 2–7 ). The samples were run on a 10% gel and transferred to a PVDF membrane. Western blotting was performed using specific Egr-1 or KSHV RTA Abs. Abs targeting β-actin were used as internal controls. ( B ) Analysis of ORF50 and egr-1 transcription. KSHV-infected HEK293 cells were lysed and RNA was extracted. Next, cDNA was synthesized and subjected to quantitative real-time PCR analysis. Baseline expression of genes at 1 hPI was considered as 1-fold for comparisons. Each point denotes the average±S.D. ( error bars ) of three experiments.

    Techniques Used: Infection, Incubation, Expressing, SDS Page, Western Blot, Synthesized, Real-time Polymerase Chain Reaction

    ( A ) Elevated Egr-1 was observed in cells transfected with egr-1/pCDNA3.1(+). BCBL-1 cells were untransfected, mock transfected, transfected with pcDNA3.1 or egr-1 /pcDNA3.1. The cells to be used as controls ( lanes 1–3 ) were incubated at 37°C for 48 h while the cells transfected with egr-1 /pcDNA3.1 were incubated for 24, 48, and 72 h, respectively. At the end of incubation, the cells were lysed and the lysates were used to perform Western blotting. ( B ) Effect of elevated Egr-1 on KSHV ORF50 and ORF8 expression. BCBL-1 cells were untransfected or transfected as described above. RNA was extracted, cDNA was synthesized, and the expression of cellular egr-1 ; and KSHV encoded ORF50 and ORF8 were analyzed by qRT-PCR. Baseline expression of genes was considered as 1-fold for comparisons. Each point denotes the average±S.D. ( error bars ) of three experiments. ( C ) Expression of lytic proteins in BCBL-1 cells transfected with Egr-1. KSHV-infected cells were untransfected, transfected with pcDNA3.1, or egr-1 /pcDNA3.1 for 48 h. The stained cells examined using a confocal microscope (magnification ×62). The average number of fluorescent cells were counted over five random fields and used for comparisons. ( D ) Enhanced egr-1 activates MAPK signaling in BCBL-1 cells. KSHV-infected cells were untransfected, mock-transfected, transfected with pcDNA3.1(+), or egr-1 /pcDNA3.1(+) for 48 h. Each group of cells was left untreated or they were treated with 10 µM of U0126 1 h prior to transfection and remained throughout the incubation period. Cell lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. ( E ) U0126 inhibits phosho-ERK1/2 and Egr-1. Briefly, BCBL-1 cells were treated with different concentrations of U0126. Following 24 h incubation, the cells were lysed and the lysates were used to perform Western blotting as per earlier protocols using specific antibodies.
    Figure Legend Snippet: ( A ) Elevated Egr-1 was observed in cells transfected with egr-1/pCDNA3.1(+). BCBL-1 cells were untransfected, mock transfected, transfected with pcDNA3.1 or egr-1 /pcDNA3.1. The cells to be used as controls ( lanes 1–3 ) were incubated at 37°C for 48 h while the cells transfected with egr-1 /pcDNA3.1 were incubated for 24, 48, and 72 h, respectively. At the end of incubation, the cells were lysed and the lysates were used to perform Western blotting. ( B ) Effect of elevated Egr-1 on KSHV ORF50 and ORF8 expression. BCBL-1 cells were untransfected or transfected as described above. RNA was extracted, cDNA was synthesized, and the expression of cellular egr-1 ; and KSHV encoded ORF50 and ORF8 were analyzed by qRT-PCR. Baseline expression of genes was considered as 1-fold for comparisons. Each point denotes the average±S.D. ( error bars ) of three experiments. ( C ) Expression of lytic proteins in BCBL-1 cells transfected with Egr-1. KSHV-infected cells were untransfected, transfected with pcDNA3.1, or egr-1 /pcDNA3.1 for 48 h. The stained cells examined using a confocal microscope (magnification ×62). The average number of fluorescent cells were counted over five random fields and used for comparisons. ( D ) Enhanced egr-1 activates MAPK signaling in BCBL-1 cells. KSHV-infected cells were untransfected, mock-transfected, transfected with pcDNA3.1(+), or egr-1 /pcDNA3.1(+) for 48 h. Each group of cells was left untreated or they were treated with 10 µM of U0126 1 h prior to transfection and remained throughout the incubation period. Cell lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. ( E ) U0126 inhibits phosho-ERK1/2 and Egr-1. Briefly, BCBL-1 cells were treated with different concentrations of U0126. Following 24 h incubation, the cells were lysed and the lysates were used to perform Western blotting as per earlier protocols using specific antibodies.

    Techniques Used: Transfection, Incubation, Western Blot, Expressing, Synthesized, Quantitative RT-PCR, Infection, Staining, Microscopy, SDS Page

    ( A ) Resveratrol inhibited egr-1 expression by as early as 6 hPI in HEK293 cells. HEK293 cells were infected with 5 MOI of KSHV for 2 h at 37°C. After infection, the cells were washed and cultured in growth medium in the presence or absence of different concentrations of resveratrol for 4 h. At the end of incubation, the cells were lysed, RNA was extracted, cDNA was synthesized, and egr-1 levels were analyzed by qRT-PCR. Each point denotes the average±S.D. ( error bars ) of three experiments. ( B ) Resveratrol inhibits MAPK activity during de novo KSHV infection. HEK293 cells were mock-infected or infected with 5 MOI of KSHV for 2 h at 37°C. After infection, the cells were washed and cultured in growth medium in the presence or absence of 100 µM of resveratrol for 48 h. The cells were lysed using gold lysis buffer (GLB) and the lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies.
    Figure Legend Snippet: ( A ) Resveratrol inhibited egr-1 expression by as early as 6 hPI in HEK293 cells. HEK293 cells were infected with 5 MOI of KSHV for 2 h at 37°C. After infection, the cells were washed and cultured in growth medium in the presence or absence of different concentrations of resveratrol for 4 h. At the end of incubation, the cells were lysed, RNA was extracted, cDNA was synthesized, and egr-1 levels were analyzed by qRT-PCR. Each point denotes the average±S.D. ( error bars ) of three experiments. ( B ) Resveratrol inhibits MAPK activity during de novo KSHV infection. HEK293 cells were mock-infected or infected with 5 MOI of KSHV for 2 h at 37°C. After infection, the cells were washed and cultured in growth medium in the presence or absence of 100 µM of resveratrol for 48 h. The cells were lysed using gold lysis buffer (GLB) and the lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies.

    Techniques Used: Expressing, Infection, Cell Culture, Incubation, Synthesized, Quantitative RT-PCR, Activity Assay, Lysis, SDS Page, Western Blot

    ( A ) RDS lowers Egr-1 and KSHV RTA expression. KSHV-infected BCBL-1 cells were synchronized in S phase and untreated or treated using 20 ng/ml of TPA for 2 h. Each group of cells was left untreated or was further treated using filtered or unfiltered RDS containing 100 µM of resveratrol. The cells were incubated at 37°C for 6 h and lysed. The lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. ( B ) RDS reduces the number of KSHV-infected cells undergoing reactivation in HEK293 cells. Mock-infected, KSHV-infected, and KSHV-infected cells in the presence of RDS containing 100 µM resveratrol were stained using monoclonal mouse anti-Egr-1 IgGs and rabbit peptide antibodies targeting KSHV RTA and examined under a fluorescent microscope (magnification ×100). ( C ) Overexpression of Egr-1 is unable to overcome RDS-mediated inhibition of virus reactivation. BCBL-1 cells were transiently transfected using pcDNA3.1(+) or egr-1 /pcDNA3.1(+) and subsequently treated with unfiltered RDS containing 100 µM of Resveratrol for 6 h. RNA was extracted, cDNA was synthesized, and egr-1 and KSHV ORF50 were analyzed by qRT-PCR. Each point denotes the average±S.D. ( error bars ) of three experiments. ( D ) RDS lowers egr-1 and KSHV ORF50 transcriptional activity. BCBL-1 cells were synchronized in S phase, treated with 20 ng/ml of TPA, and treated using filtered or unfiltered RDS containing 100 µM of resveratrol as before. RNA was extracted, cDNA was synthesized, and egr-1 and KSHV ORF50 were analyzed by qRT-PCR. Each point denotes the average±S.D. ( error bars ) of three experiments. Columns with different alphabets are statistically significant (P<0.05) by LSD. ( E ) RDS inhibits the ability of Egr-1 to bind KSHV ORF50 in vivo . BCBL-1 cells were synchronized and treated with TPA as before. The cells were further treated using unfiltered RDS containing 100 µM of resveratrol and incubated for 6 h. ChIP assays were performed using 2 µg of specific Egr-1 Abs. Semi-quantitative PCR experiments were performed using samples from 1% input DNA and IP samples in order to determine the expression of ORF50 P8. Respective cDNA at 10, 15, 20, 25, and 30 cycles were resolved on a 2% agarose gel. Specific antibodies to histone H3 and nonspecific IgGs were used to IP sample chromatin and served as positive and negative controls, respectively.
    Figure Legend Snippet: ( A ) RDS lowers Egr-1 and KSHV RTA expression. KSHV-infected BCBL-1 cells were synchronized in S phase and untreated or treated using 20 ng/ml of TPA for 2 h. Each group of cells was left untreated or was further treated using filtered or unfiltered RDS containing 100 µM of resveratrol. The cells were incubated at 37°C for 6 h and lysed. The lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. ( B ) RDS reduces the number of KSHV-infected cells undergoing reactivation in HEK293 cells. Mock-infected, KSHV-infected, and KSHV-infected cells in the presence of RDS containing 100 µM resveratrol were stained using monoclonal mouse anti-Egr-1 IgGs and rabbit peptide antibodies targeting KSHV RTA and examined under a fluorescent microscope (magnification ×100). ( C ) Overexpression of Egr-1 is unable to overcome RDS-mediated inhibition of virus reactivation. BCBL-1 cells were transiently transfected using pcDNA3.1(+) or egr-1 /pcDNA3.1(+) and subsequently treated with unfiltered RDS containing 100 µM of Resveratrol for 6 h. RNA was extracted, cDNA was synthesized, and egr-1 and KSHV ORF50 were analyzed by qRT-PCR. Each point denotes the average±S.D. ( error bars ) of three experiments. ( D ) RDS lowers egr-1 and KSHV ORF50 transcriptional activity. BCBL-1 cells were synchronized in S phase, treated with 20 ng/ml of TPA, and treated using filtered or unfiltered RDS containing 100 µM of resveratrol as before. RNA was extracted, cDNA was synthesized, and egr-1 and KSHV ORF50 were analyzed by qRT-PCR. Each point denotes the average±S.D. ( error bars ) of three experiments. Columns with different alphabets are statistically significant (P<0.05) by LSD. ( E ) RDS inhibits the ability of Egr-1 to bind KSHV ORF50 in vivo . BCBL-1 cells were synchronized and treated with TPA as before. The cells were further treated using unfiltered RDS containing 100 µM of resveratrol and incubated for 6 h. ChIP assays were performed using 2 µg of specific Egr-1 Abs. Semi-quantitative PCR experiments were performed using samples from 1% input DNA and IP samples in order to determine the expression of ORF50 P8. Respective cDNA at 10, 15, 20, 25, and 30 cycles were resolved on a 2% agarose gel. Specific antibodies to histone H3 and nonspecific IgGs were used to IP sample chromatin and served as positive and negative controls, respectively.

    Techniques Used: Expressing, Infection, Incubation, SDS Page, Western Blot, Staining, Microscopy, Over Expression, Inhibition, Transfection, Synthesized, Quantitative RT-PCR, Activity Assay, In Vivo, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis

    egr1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr1
    Upper panel: triplicate sets of each cell line were treated with or without PDGF-BB for 1 h. mRNA expression profiles for the probe sets identified as differentially expressed in the treatment group for all of the cell lines, except for double knockout. Expression levels of the probe sets interrogating the genes that were commonly differentially expressed in the PDGFRβ−/− (beta null), PDGFRα−/− (alpha null), and WT (PDGFRα+/+ PDGFRβ+/+) cell lines are shown in the heatmap. Each column represents a sample, each row a gene. Column labels indicate the cell line and treatment condition (T for samples treated with PDGF-BB, U for untreated samples) for each sample. Each probe set's expression has been independently normalized across the experiments. Bright green shading indicates an expression level below the gene-wise mean, bright red indicates an expression level above the mean, while darker shades indicate expression levels closer to the mean intensity. Lower panel: four cell lines were treated with PDGF-BB (50 ng/ml) for various times and their lysates were immunoblotted for ATF-3, Txnip, Fra-1, Nurr1, Nur77, TF, and <t>EGR1</t> (PDGF treatment does not influence the total Erk, therefore Erk was regarded as a spotting control in this system, the same for the following experiments in the MEF cell lines with genetically defined PDGFRs) to validate findings from the mRNA expression analysis (upper panel). Expression levels of probe sets interrogating the genes for these proteins are shown in the heatmap above the western blot figure.
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    1) Product Images from "Comprehensive Dissection of PDGF-PDGFR Signaling Pathways in PDGFR Genetically Defined Cells"

    Article Title: Comprehensive Dissection of PDGF-PDGFR Signaling Pathways in PDGFR Genetically Defined Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0003794

    Upper panel: triplicate sets of each cell line were treated with or without PDGF-BB for 1 h. mRNA expression profiles for the probe sets identified as differentially expressed in the treatment group for all of the cell lines, except for double knockout. Expression levels of the probe sets interrogating the genes that were commonly differentially expressed in the PDGFRβ−/− (beta null), PDGFRα−/− (alpha null), and WT (PDGFRα+/+ PDGFRβ+/+) cell lines are shown in the heatmap. Each column represents a sample, each row a gene. Column labels indicate the cell line and treatment condition (T for samples treated with PDGF-BB, U for untreated samples) for each sample. Each probe set's expression has been independently normalized across the experiments. Bright green shading indicates an expression level below the gene-wise mean, bright red indicates an expression level above the mean, while darker shades indicate expression levels closer to the mean intensity. Lower panel: four cell lines were treated with PDGF-BB (50 ng/ml) for various times and their lysates were immunoblotted for ATF-3, Txnip, Fra-1, Nurr1, Nur77, TF, and EGR1 (PDGF treatment does not influence the total Erk, therefore Erk was regarded as a spotting control in this system, the same for the following experiments in the MEF cell lines with genetically defined PDGFRs) to validate findings from the mRNA expression analysis (upper panel). Expression levels of probe sets interrogating the genes for these proteins are shown in the heatmap above the western blot figure.
    Figure Legend Snippet: Upper panel: triplicate sets of each cell line were treated with or without PDGF-BB for 1 h. mRNA expression profiles for the probe sets identified as differentially expressed in the treatment group for all of the cell lines, except for double knockout. Expression levels of the probe sets interrogating the genes that were commonly differentially expressed in the PDGFRβ−/− (beta null), PDGFRα−/− (alpha null), and WT (PDGFRα+/+ PDGFRβ+/+) cell lines are shown in the heatmap. Each column represents a sample, each row a gene. Column labels indicate the cell line and treatment condition (T for samples treated with PDGF-BB, U for untreated samples) for each sample. Each probe set's expression has been independently normalized across the experiments. Bright green shading indicates an expression level below the gene-wise mean, bright red indicates an expression level above the mean, while darker shades indicate expression levels closer to the mean intensity. Lower panel: four cell lines were treated with PDGF-BB (50 ng/ml) for various times and their lysates were immunoblotted for ATF-3, Txnip, Fra-1, Nurr1, Nur77, TF, and EGR1 (PDGF treatment does not influence the total Erk, therefore Erk was regarded as a spotting control in this system, the same for the following experiments in the MEF cell lines with genetically defined PDGFRs) to validate findings from the mRNA expression analysis (upper panel). Expression levels of probe sets interrogating the genes for these proteins are shown in the heatmap above the western blot figure.

    Techniques Used: Expressing, Double Knockout, Western Blot

    A and B: Triplicate sets of each cell line were treated with or without PDGF-BB (50 ng/ml) for 1 h. Expression levels of the genes in the IL6 pathway in the WT cell line are shown in panel A. Expression values are summarized over multiple probe sets for each gene, and a standard Student's T-test p-value indicates the strength of the difference-of-the-means between the treatment groups. In panel B, the expression patterns (across all cell lines) of a few selected genes from the IL6 pathway are shown along with their respective Western blot results in panel C. In both heatmaps, bright green shading indicates an expression level below the gene-wise mean, bright red indicates an expression level above the mean, while darker shades indicate expression levels closer to the mean intensity. The mean and standard deviation for each (log-reduced) gene are shown to the right of each gene name. C: Four cell lines were treated with PDGF-BB (50 ng/ml) for 10 min, 1 h. Cell lysates were immunoblotted for Jun, Cebpb, Fos and ERK (lower panel). D: Four cell lines were pretreated with STI-571 (5 µM) for 90 minutes and then stimulated with PDGF-BB (50 ng/ml) for 1 h. The harvested lysates were immunoblotted for PDGFRα, PDGFRβ, AKT, p-AKT (Ser473), p-ERK (Tyr 204), ERK, EGR1, c-Jun, c-Fos, ATF-3, Fra-1, and β-actin (β-actin as internal control).
    Figure Legend Snippet: A and B: Triplicate sets of each cell line were treated with or without PDGF-BB (50 ng/ml) for 1 h. Expression levels of the genes in the IL6 pathway in the WT cell line are shown in panel A. Expression values are summarized over multiple probe sets for each gene, and a standard Student's T-test p-value indicates the strength of the difference-of-the-means between the treatment groups. In panel B, the expression patterns (across all cell lines) of a few selected genes from the IL6 pathway are shown along with their respective Western blot results in panel C. In both heatmaps, bright green shading indicates an expression level below the gene-wise mean, bright red indicates an expression level above the mean, while darker shades indicate expression levels closer to the mean intensity. The mean and standard deviation for each (log-reduced) gene are shown to the right of each gene name. C: Four cell lines were treated with PDGF-BB (50 ng/ml) for 10 min, 1 h. Cell lysates were immunoblotted for Jun, Cebpb, Fos and ERK (lower panel). D: Four cell lines were pretreated with STI-571 (5 µM) for 90 minutes and then stimulated with PDGF-BB (50 ng/ml) for 1 h. The harvested lysates were immunoblotted for PDGFRα, PDGFRβ, AKT, p-AKT (Ser473), p-ERK (Tyr 204), ERK, EGR1, c-Jun, c-Fos, ATF-3, Fra-1, and β-actin (β-actin as internal control).

    Techniques Used: Expressing, Western Blot, Standard Deviation

    egr1 ab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr1 ab
    Egr1 Ab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti egr1 15f7 cs 4351
    HGF-induced activation of <t>Egr1</t> mRNA and protein level ( 2A, 2C ) and the effect of heparin on the HGF induced Egr1 expression in SK-HEP-1 cells were examined by RT-PCR and western blotting ( 2B, 2D ). Egr1 transcriptional activity was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with pGL2-Luc-B-Egr1 plasmid construct and the pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity ( 2E, 2F ). Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.
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    Cell Signaling Technology Inc anti egr 1
    Protocols of experimental grouping and reagent administering. (a) Protocol used to investigate role of ERK1/2 and <t>Egr-1</t> in extracellular-calcium-containing-H/R injury. (b) Protocol used to investigate role of PKC α /ERK1/2/Egr-1 in extracellular-calcium-containing-H/R injury. (c) Protocol used to investigate role of ERK1/2 and Egr-1 in extracellular-calcium-free-H/R injury. (d) Protocol used to investigate role of cAMP/PKA in extracellular-calcium-free-H/R injury. CaCon, calcium-containing normoxia; CaH/R, calcium-containing H/R; 0CaCon, calcium-free normoxic control; 0CaH/R, calcium-free H/R.
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    Cell Signaling Technology Inc rabbit anti egr 1 antibody
    (A) HMEC-1s and HUVECs were incubated with SW480-derived EVs (1 µg/mL) or untreated control. mRNA was isolated from untreated control cells or cells treated with EVs for 0, 0.5, 1, 2, and 4 h and analyzed using real time RT-PCR (n = 3). Values represent <t>Egr-1</t> mRNA/GAPDH mRNA normalized to untreated control cells. (B) HMEC-1s were treated with EVs (1 µg/mL) derived from HCT116 colorectal carcinoma, A549 lung adenocarcinoma, HT1080 fibrosarcoma, PC3 prostate carcinoma, SH-SY5Y neuroblastoma, and BEAS-2B normal bronchial epithelial cells for 0.5 h (n = 3). (C, D) In HMEC-1s, nuclear translocation of Egr-1 protein after stimulation with SW480-derived EVs (1 µg/mL) for 0.5, 1, 2, and 4 h was analyzed under confocal microscopy (n = 3). Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. Co-localized fluorescence signals (purple) indicate the translocation of Egr-1 into the nucleus. Representative photographs are shown in panel C. Scale bars represent 30 µm. The percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus colocalized signals over that of total cells (D). Data are represented as mean ± SD. * P <0.05; ** P <0.01; ** P <0.001; n.s., not significant.
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    Cell Signaling Technology Inc egr1 primary antibody
    A. WT1 mRNA (+KTS and –KTS) splice variant PCR products expressed in LβT2 cells. Whole cell RNA was extracted from LβT2 cells, reverse transcribed to cDNA and quantified by real-time PCR, using specific primers to detect +KTS and –KTS splice variants, then displayed on a 1% agarose gel. Bands corresponding to the products for +KTS (301bp) and –KTS (292 bp) WT1 were detected in 4 independent samples of mRNA. B. WT1 and <t>Egr1</t> protein expression in LβT2 cells. Cell proteins (30μg) were separated on 10% polyacrylamide-SDS gels, then analyzed by immunoblotting with specific antibodies for WT1, Egr1 and β-actin. Specific proteins were detected in the same samples of untreated LβT2 cells. C. Chromatin association of WT1 with the endogenous LHβ promoter in LβT2 cells. The association of WT1 and RNA Polymerase II with the LHβ promoter in untreated LβT2 cells was measured by Chromatin immunoprecipitation assays with antibodies against WT1 and phosphorylated RNApol II, as well as control (no Antibody). LHβ promoter occupancy was measured by quantitative real time PCR using primers specific for the LHβ promoter, and normalized for chromatin input in each sample. In this study, background binding (no Antibody) was set at 100% and association of RNA Polymerase II and WT1 are expressed relative to background values. Association was measured in 3 independent experiments with duplicate samples.
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    Cell Signaling Technology Inc rabbit monoclonal antibodies egr
    Growth-quiescent SMCs were treated with MMP inhibitor GM6001+ (25 µM) and TAPI-1 (10 µM) for 30 min before stimulation with IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). ( A ) <t>Egr-1</t> mRNA levels in GM6001+ or GM6001- treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( B ) Western blotting for Egr-1 or beta-actin in total extracts of SMCs treated with GM6001+ or GM6001-. ( C ) Egr-1 mRNA levels in TAPI-1 treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( D ) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with TAPI-1. DMSO was used as a carrier. ( E ) Western blotting for ADAM17, ADAM10, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ADAM17 siRNA (0.1 µM) and treated with IL-1beta for 30 min.
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    Cell Signaling Technology Inc rabbit monoclonal anti egr1 antibody
    Oligonucleotides and plasmids.
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    Cell Signaling Technology Inc egr1
    Oligonucleotides and plasmids.
    Egr1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc egr 1
    ( A ) IVT-synthesized <t>Egr-1</t> binds to ORF50 P probes. EMSA studies were performed using IVT-synthesized Egr-1 products and DIG-labeled ORF50 P probes (see ). ( B ) Mutations in the putative Egr-1 binding domain inhibit Egr-1 binding. EMSA experiments were performed using wt ORF50 P3 and ORF50 P8 probes as well as corresponding probes displaying mutations in the suspected Egr-1 binding domain ( ORF50 P3m and ORF50 P8m). ( C ) Nuclear lysates from KSHV-infected cells formed a complex with ORF50 P probes. BCBL-1 cells were synchronized in S phase of cell cycle according to earlier protocols , treated with 20 ng/ml TPA for 8 h, and lysed. Nuclear extracts containing Egr-1 proteins were used to perform EMSA studies using ORF50 P3 and ORF50 P8 probes. Specific Egr-1 binding was confirmed by performing supershifts using specific antibodies to Egr-1 ( lanes 4 and 8 ) or nonspecific IgGs ( lanes 3 and 7 ). The arrowhead indicates protein/DNA complex formation. Specific antibody/protein/DNA supershifts are denoted by the asterisk.
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    Cell Signaling Technology Inc egr1 ab
    ( A ) IVT-synthesized <t>Egr-1</t> binds to ORF50 P probes. EMSA studies were performed using IVT-synthesized Egr-1 products and DIG-labeled ORF50 P probes (see ). ( B ) Mutations in the putative Egr-1 binding domain inhibit Egr-1 binding. EMSA experiments were performed using wt ORF50 P3 and ORF50 P8 probes as well as corresponding probes displaying mutations in the suspected Egr-1 binding domain ( ORF50 P3m and ORF50 P8m). ( C ) Nuclear lysates from KSHV-infected cells formed a complex with ORF50 P probes. BCBL-1 cells were synchronized in S phase of cell cycle according to earlier protocols , treated with 20 ng/ml TPA for 8 h, and lysed. Nuclear extracts containing Egr-1 proteins were used to perform EMSA studies using ORF50 P3 and ORF50 P8 probes. Specific Egr-1 binding was confirmed by performing supershifts using specific antibodies to Egr-1 ( lanes 4 and 8 ) or nonspecific IgGs ( lanes 3 and 7 ). The arrowhead indicates protein/DNA complex formation. Specific antibody/protein/DNA supershifts are denoted by the asterisk.
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    Image Search Results


    HGF-induced activation of Egr1 mRNA and protein level ( 2A, 2C ) and the effect of heparin on the HGF induced Egr1 expression in SK-HEP-1 cells were examined by RT-PCR and western blotting ( 2B, 2D ). Egr1 transcriptional activity was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with pGL2-Luc-B-Egr1 plasmid construct and the pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity ( 2E, 2F ). Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

    Journal: PLoS ONE

    Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

    doi: 10.1371/journal.pone.0042717

    Figure Lengend Snippet: HGF-induced activation of Egr1 mRNA and protein level ( 2A, 2C ) and the effect of heparin on the HGF induced Egr1 expression in SK-HEP-1 cells were examined by RT-PCR and western blotting ( 2B, 2D ). Egr1 transcriptional activity was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with pGL2-Luc-B-Egr1 plasmid construct and the pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity ( 2E, 2F ). Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

    Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Construct

    HGF-induced activation of Egr1 and the effect of c-Met inhibition on HGF-induced Egr1 expression in SK-HEP-1 cells were examined by immunoblotting and luciferase reporter assays ( 5A, 5B ). Transcriptional activity of Egr1 was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with a pGL2-Luc-B-Egr1 plasmid construct and pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity. Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

    Journal: PLoS ONE

    Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

    doi: 10.1371/journal.pone.0042717

    Figure Lengend Snippet: HGF-induced activation of Egr1 and the effect of c-Met inhibition on HGF-induced Egr1 expression in SK-HEP-1 cells were examined by immunoblotting and luciferase reporter assays ( 5A, 5B ). Transcriptional activity of Egr1 was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with a pGL2-Luc-B-Egr1 plasmid construct and pRL-TK Renilia luciferase. Relative luciferase activity was determined as described in . The firefly luciferase activity was normalized to Renilia luciferase activity. Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

    Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, Inhibition, Expressing, Western Blot, Luciferase, Activity Assay, Reporter Assay, Transfection, Plasmid Preparation, Construct

    To achieve an inducible knockdown of Egr1, we firstly transfected SK-HEP-1 cells with pCDNA6/TR which express tet repressor. Stable cells were selected using Blasticidin before co-transfecting SK-HEP-1 T-REx cells with tetracycline–regulated pSUPER.retro.neo+GFP tet RNAi construct, which was directed against a sequence containing Egr1. Stable cells were selected using G418. After selection, clones were analyzed for downregulation of Egr1 after 24 h of tetracycline treatment. Ten-fold down-regulation of Egr1 was detected by immunoblotting in pSUPER.retro.neo+GFP tet/Egr1 clones ( 6A ) but not in mock transfected clones ( 6B ). The graph compares the signal intensities obtained from Egr1 bands.

    Journal: PLoS ONE

    Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

    doi: 10.1371/journal.pone.0042717

    Figure Lengend Snippet: To achieve an inducible knockdown of Egr1, we firstly transfected SK-HEP-1 cells with pCDNA6/TR which express tet repressor. Stable cells were selected using Blasticidin before co-transfecting SK-HEP-1 T-REx cells with tetracycline–regulated pSUPER.retro.neo+GFP tet RNAi construct, which was directed against a sequence containing Egr1. Stable cells were selected using G418. After selection, clones were analyzed for downregulation of Egr1 after 24 h of tetracycline treatment. Ten-fold down-regulation of Egr1 was detected by immunoblotting in pSUPER.retro.neo+GFP tet/Egr1 clones ( 6A ) but not in mock transfected clones ( 6B ). The graph compares the signal intensities obtained from Egr1 bands.

    Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Transfection, Construct, Sequencing, Selection, Clone Assay, Western Blot

    SK-HEP-1 TREx cells were obtained by stable transfection of pCDNA-6/TR. Then SK-HEP-1 TREx cells were transfected with recombinant pSUPER-retro.neo+GFP vector expressing Egr-1 targeted shRNA or empty pSUPER-retro.neo+GFP vector using Fugene as described in . Knockdown of Egr1 in Egr1-shRNA-expressing SK-HEP-1 stable cell lines were induced by tetracycline ( 7A ). Equal amount of tetracycline induced and un-induced Egr1-shRNA-expressing SK-HEP-1 cells were used for migration and invasion assays in the presence and absence of HGF ( 7B, 7C ). Results are expressed as fold differences of matrigel invaded cells and are representative of two or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups. Zymographic gel showing active MMP-2 and-9 bands in 24 h conditioned medium of cultured Egr1-shRNA-expressing SK-HEP-1 cells left untreated or stimulated with tetracycline and/or HGF ( 7D, 7E ). Protein levels of MT1-MMP were studied in tetracyclin and/or HGF-induced SK-HEP-1-Egr1 cells ( 7F ). Densitometric analysis of gelatin zymography and immunoblots showed that HGF induces MT1-MMP expression and MMP-2 and -9 activation, whereas silencing of Egr1 blocks HGF-induced MMPs up-regulation.

    Journal: PLoS ONE

    Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

    doi: 10.1371/journal.pone.0042717

    Figure Lengend Snippet: SK-HEP-1 TREx cells were obtained by stable transfection of pCDNA-6/TR. Then SK-HEP-1 TREx cells were transfected with recombinant pSUPER-retro.neo+GFP vector expressing Egr-1 targeted shRNA or empty pSUPER-retro.neo+GFP vector using Fugene as described in . Knockdown of Egr1 in Egr1-shRNA-expressing SK-HEP-1 stable cell lines were induced by tetracycline ( 7A ). Equal amount of tetracycline induced and un-induced Egr1-shRNA-expressing SK-HEP-1 cells were used for migration and invasion assays in the presence and absence of HGF ( 7B, 7C ). Results are expressed as fold differences of matrigel invaded cells and are representative of two or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups. Zymographic gel showing active MMP-2 and-9 bands in 24 h conditioned medium of cultured Egr1-shRNA-expressing SK-HEP-1 cells left untreated or stimulated with tetracycline and/or HGF ( 7D, 7E ). Protein levels of MT1-MMP were studied in tetracyclin and/or HGF-induced SK-HEP-1-Egr1 cells ( 7F ). Densitometric analysis of gelatin zymography and immunoblots showed that HGF induces MT1-MMP expression and MMP-2 and -9 activation, whereas silencing of Egr1 blocks HGF-induced MMPs up-regulation.

    Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Stable Transfection, Transfection, Recombinant, Plasmid Preparation, Expressing, shRNA, Migration, Cell Culture, Zymography, Western Blot, Activation Assay

    The effect of Egr1 on the cell invasion of HCC cell lines were examined using SNU-449 transiently transfected with pCMV-6-AC-GFP Egr1 overexpression vector ( 8A ). Equal amounts of wild type and Egr1 overexpressing SNU-449 cells were used for migration ( 8B ) and invasion ( 8C ) assay in the presence and absence of HGF. Results are expressed as fold differences of matrigel invaded cells. Zymographic gel showing the active MMP-9 bands in 24 h conditioned medium of cultured Egr1-overexpressing SNU-449 cells ( 8D ). The graph compares the signal intensities obtained from zymography gels.

    Journal: PLoS ONE

    Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

    doi: 10.1371/journal.pone.0042717

    Figure Lengend Snippet: The effect of Egr1 on the cell invasion of HCC cell lines were examined using SNU-449 transiently transfected with pCMV-6-AC-GFP Egr1 overexpression vector ( 8A ). Equal amounts of wild type and Egr1 overexpressing SNU-449 cells were used for migration ( 8B ) and invasion ( 8C ) assay in the presence and absence of HGF. Results are expressed as fold differences of matrigel invaded cells. Zymographic gel showing the active MMP-9 bands in 24 h conditioned medium of cultured Egr1-overexpressing SNU-449 cells ( 8D ). The graph compares the signal intensities obtained from zymography gels.

    Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Transfection, Over Expression, Plasmid Preparation, Migration, Cell Culture, Zymography

    The effects of heparin and HGF on Egr1 expression in Egr1 over-expressing SNU-449 cells was analyzed by immunoblotting ( 9A ). Following serum deprivation, Egr1 overexpressed SN-449 cells were left untreated (0) or treated with HGF in the presence or absence of heparin, then invasion and migration assays were performed using the Roche xCELLigence System ( 9B, 9C ). Results are representative of three or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

    Journal: PLoS ONE

    Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

    doi: 10.1371/journal.pone.0042717

    Figure Lengend Snippet: The effects of heparin and HGF on Egr1 expression in Egr1 over-expressing SNU-449 cells was analyzed by immunoblotting ( 9A ). Following serum deprivation, Egr1 overexpressed SN-449 cells were left untreated (0) or treated with HGF in the presence or absence of heparin, then invasion and migration assays were performed using the Roche xCELLigence System ( 9B, 9C ). Results are representative of three or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.

    Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Western Blot, Migration

    Activation of HGF/c-Met signaling in HCC cells leads to MAPK signaling (i), and up-regulation of Egr1 (ii), which in turn induces MMP-2 and MMP-9 activation and MT1-MMP expression (iii), and increased cell motility and invasion (iv). Heparin treatment results in the inhibition of HGF-induced cellular invasion via repression of HGF-induced c-Met activation, as well as inhibition of MAPK signaling, downregulation of Egr1 expression, and MMP activation (v).

    Journal: PLoS ONE

    Article Title: Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

    doi: 10.1371/journal.pone.0042717

    Figure Lengend Snippet: Activation of HGF/c-Met signaling in HCC cells leads to MAPK signaling (i), and up-regulation of Egr1 (ii), which in turn induces MMP-2 and MMP-9 activation and MT1-MMP expression (iii), and increased cell motility and invasion (iv). Heparin treatment results in the inhibition of HGF-induced cellular invasion via repression of HGF-induced c-Met activation, as well as inhibition of MAPK signaling, downregulation of Egr1 expression, and MMP activation (v).

    Article Snippet: Anti-Egr1 (15F7) cs 4351, Anti-phosho-Met (Y-1234/1235) cs 3129 and anti phosho-44/22 MAPK (Thr202/Tyr204) (19G2) 4377 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, Expressing, Inhibition

    Protocols of experimental grouping and reagent administering. (a) Protocol used to investigate role of ERK1/2 and Egr-1 in extracellular-calcium-containing-H/R injury. (b) Protocol used to investigate role of PKC α /ERK1/2/Egr-1 in extracellular-calcium-containing-H/R injury. (c) Protocol used to investigate role of ERK1/2 and Egr-1 in extracellular-calcium-free-H/R injury. (d) Protocol used to investigate role of cAMP/PKA in extracellular-calcium-free-H/R injury. CaCon, calcium-containing normoxia; CaH/R, calcium-containing H/R; 0CaCon, calcium-free normoxic control; 0CaH/R, calcium-free H/R.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: N -n-Butyl Haloperidol Iodide Ameliorates Cardiomyocytes Hypoxia/Reoxygenation Injury by Extracellular Calcium-Dependent and -Independent Mechanisms

    doi: 10.1155/2013/912310

    Figure Lengend Snippet: Protocols of experimental grouping and reagent administering. (a) Protocol used to investigate role of ERK1/2 and Egr-1 in extracellular-calcium-containing-H/R injury. (b) Protocol used to investigate role of PKC α /ERK1/2/Egr-1 in extracellular-calcium-containing-H/R injury. (c) Protocol used to investigate role of ERK1/2 and Egr-1 in extracellular-calcium-free-H/R injury. (d) Protocol used to investigate role of cAMP/PKA in extracellular-calcium-free-H/R injury. CaCon, calcium-containing normoxia; CaH/R, calcium-containing H/R; 0CaCon, calcium-free normoxic control; 0CaH/R, calcium-free H/R.

    Article Snippet: Anti-p-PKC α , anti-total PKC α , anti-PKA, and chemiluminescence luminol reagents were purchased from Santa Cruz Biotechnology (U.S.); anti-p-ERK1/2, anti-total ERK1/2, and anti-Egr-1 were purchased from Cell Signaling Technology (U.S.); anti- β -actin and horseradish peroxidase-conjugated secondary antibodies were purchased from Wuhan Boster Biotechnology Limited Company (China); all the other chemicals and reagents were purchased from local agencies.

    Techniques:

    Effects of F 2 , Verapamil, and ERK1/2 inhibitors and activator on p-ERK1/2, total ERK1/2, and Egr-1 expression in extracellular-calcium-containing myocardial H/R by western-blot assay. (a) p-ERK1/2 and total ERK1/2; (b) Egr-1 protein. Quantitative densitometric data were expressed as percentages of the level observed in the CaCon group. All values are expressed as mean ± SEM of at least six individual experiments. * P < 0.05 versus CaCon group; # P < 0.05 versus CaH/R group; † P < 0.05 versus CaH/R+F 2 group.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: N -n-Butyl Haloperidol Iodide Ameliorates Cardiomyocytes Hypoxia/Reoxygenation Injury by Extracellular Calcium-Dependent and -Independent Mechanisms

    doi: 10.1155/2013/912310

    Figure Lengend Snippet: Effects of F 2 , Verapamil, and ERK1/2 inhibitors and activator on p-ERK1/2, total ERK1/2, and Egr-1 expression in extracellular-calcium-containing myocardial H/R by western-blot assay. (a) p-ERK1/2 and total ERK1/2; (b) Egr-1 protein. Quantitative densitometric data were expressed as percentages of the level observed in the CaCon group. All values are expressed as mean ± SEM of at least six individual experiments. * P < 0.05 versus CaCon group; # P < 0.05 versus CaH/R group; † P < 0.05 versus CaH/R+F 2 group.

    Article Snippet: Anti-p-PKC α , anti-total PKC α , anti-PKA, and chemiluminescence luminol reagents were purchased from Santa Cruz Biotechnology (U.S.); anti-p-ERK1/2, anti-total ERK1/2, and anti-Egr-1 were purchased from Cell Signaling Technology (U.S.); anti- β -actin and horseradish peroxidase-conjugated secondary antibodies were purchased from Wuhan Boster Biotechnology Limited Company (China); all the other chemicals and reagents were purchased from local agencies.

    Techniques: Expressing, Western Blot

    Effects of F 2 , Verapamil, and PKC α inhibitor and activator on p-PKC α , total PKC α , p-ERK1/2, total ERK1/2, and Egr-1 expression in extracellular-calcium-containing myocardial H/R by western-blot assay. (a) p-PKC α and total PKC α protein levels; (b) p-ERK1/2 and total ERK1/2 protein levels; (c) Egr-1 protein levels. All values are expressed as mean ± S.E.M. of at least six individual experiments. * P < 0.05 versus CaCon group; # P < 0.05 versus CaH/R group; † P < 0.05 versus CaH/R+F 2 group.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: N -n-Butyl Haloperidol Iodide Ameliorates Cardiomyocytes Hypoxia/Reoxygenation Injury by Extracellular Calcium-Dependent and -Independent Mechanisms

    doi: 10.1155/2013/912310

    Figure Lengend Snippet: Effects of F 2 , Verapamil, and PKC α inhibitor and activator on p-PKC α , total PKC α , p-ERK1/2, total ERK1/2, and Egr-1 expression in extracellular-calcium-containing myocardial H/R by western-blot assay. (a) p-PKC α and total PKC α protein levels; (b) p-ERK1/2 and total ERK1/2 protein levels; (c) Egr-1 protein levels. All values are expressed as mean ± S.E.M. of at least six individual experiments. * P < 0.05 versus CaCon group; # P < 0.05 versus CaH/R group; † P < 0.05 versus CaH/R+F 2 group.

    Article Snippet: Anti-p-PKC α , anti-total PKC α , anti-PKA, and chemiluminescence luminol reagents were purchased from Santa Cruz Biotechnology (U.S.); anti-p-ERK1/2, anti-total ERK1/2, and anti-Egr-1 were purchased from Cell Signaling Technology (U.S.); anti- β -actin and horseradish peroxidase-conjugated secondary antibodies were purchased from Wuhan Boster Biotechnology Limited Company (China); all the other chemicals and reagents were purchased from local agencies.

    Techniques: Expressing, Western Blot

    Effects of F 2 , ERK1/2 inhibitors, and activator on p-ERK1/2, total ERK1/2, and Egr-1 expression in extracellular-calcium-free myocardial H/R by western-blot assay. All values are expressed as mean ± S.E.M. of at least six individual experiments. * P < 0.05 versus 0CaCon group; # P < 0.05 versus 0CaH/R group; † P < 0.05 versus 0CaH/R+F 2 group.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: N -n-Butyl Haloperidol Iodide Ameliorates Cardiomyocytes Hypoxia/Reoxygenation Injury by Extracellular Calcium-Dependent and -Independent Mechanisms

    doi: 10.1155/2013/912310

    Figure Lengend Snippet: Effects of F 2 , ERK1/2 inhibitors, and activator on p-ERK1/2, total ERK1/2, and Egr-1 expression in extracellular-calcium-free myocardial H/R by western-blot assay. All values are expressed as mean ± S.E.M. of at least six individual experiments. * P < 0.05 versus 0CaCon group; # P < 0.05 versus 0CaH/R group; † P < 0.05 versus 0CaH/R+F 2 group.

    Article Snippet: Anti-p-PKC α , anti-total PKC α , anti-PKA, and chemiluminescence luminol reagents were purchased from Santa Cruz Biotechnology (U.S.); anti-p-ERK1/2, anti-total ERK1/2, and anti-Egr-1 were purchased from Cell Signaling Technology (U.S.); anti- β -actin and horseradish peroxidase-conjugated secondary antibodies were purchased from Wuhan Boster Biotechnology Limited Company (China); all the other chemicals and reagents were purchased from local agencies.

    Techniques: Expressing, Western Blot

    (A) HMEC-1s and HUVECs were incubated with SW480-derived EVs (1 µg/mL) or untreated control. mRNA was isolated from untreated control cells or cells treated with EVs for 0, 0.5, 1, 2, and 4 h and analyzed using real time RT-PCR (n = 3). Values represent Egr-1 mRNA/GAPDH mRNA normalized to untreated control cells. (B) HMEC-1s were treated with EVs (1 µg/mL) derived from HCT116 colorectal carcinoma, A549 lung adenocarcinoma, HT1080 fibrosarcoma, PC3 prostate carcinoma, SH-SY5Y neuroblastoma, and BEAS-2B normal bronchial epithelial cells for 0.5 h (n = 3). (C, D) In HMEC-1s, nuclear translocation of Egr-1 protein after stimulation with SW480-derived EVs (1 µg/mL) for 0.5, 1, 2, and 4 h was analyzed under confocal microscopy (n = 3). Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. Co-localized fluorescence signals (purple) indicate the translocation of Egr-1 into the nucleus. Representative photographs are shown in panel C. Scale bars represent 30 µm. The percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus colocalized signals over that of total cells (D). Data are represented as mean ± SD. * P <0.05; ** P <0.01; ** P <0.001; n.s., not significant.

    Journal: PLoS ONE

    Article Title: Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

    doi: 10.1371/journal.pone.0115170

    Figure Lengend Snippet: (A) HMEC-1s and HUVECs were incubated with SW480-derived EVs (1 µg/mL) or untreated control. mRNA was isolated from untreated control cells or cells treated with EVs for 0, 0.5, 1, 2, and 4 h and analyzed using real time RT-PCR (n = 3). Values represent Egr-1 mRNA/GAPDH mRNA normalized to untreated control cells. (B) HMEC-1s were treated with EVs (1 µg/mL) derived from HCT116 colorectal carcinoma, A549 lung adenocarcinoma, HT1080 fibrosarcoma, PC3 prostate carcinoma, SH-SY5Y neuroblastoma, and BEAS-2B normal bronchial epithelial cells for 0.5 h (n = 3). (C, D) In HMEC-1s, nuclear translocation of Egr-1 protein after stimulation with SW480-derived EVs (1 µg/mL) for 0.5, 1, 2, and 4 h was analyzed under confocal microscopy (n = 3). Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. Co-localized fluorescence signals (purple) indicate the translocation of Egr-1 into the nucleus. Representative photographs are shown in panel C. Scale bars represent 30 µm. The percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus colocalized signals over that of total cells (D). Data are represented as mean ± SD. * P <0.05; ** P <0.01; ** P <0.001; n.s., not significant.

    Article Snippet: Cells were treated with SW480-derived EVs (1 µg/mL, 0.5 mL), fixed with 4% paraformaldehyde, and incubated with rabbit anti-Egr-1 antibody (Cell Signaling Technology, Hitchin, United Kingdom) followed by AlexaFluor488 goat anti-rabbit IgG antibody (Invitrogen).

    Techniques: Incubation, Derivative Assay, Isolation, Quantitative RT-PCR, Translocation Assay, Confocal Microscopy, Staining, Fluorescence

    (A) HMEC-1s were transfected with 50 nM of scrambled siRNA or Egr-1 siRNA-1, Egr-1 siRNA-2, or Egr-1 siRNA-3. mRNAs were isolated from the cells after 48 h and the level of Egr-1 mRNA was analyzed using real time RT-PCR (n = 3). (B) Nuclear translocation of Egr-1 protein after stimulation with SW480-derived EVs (1 µg/mL) for 1 h was analyzed in scrambled siRNA (50 nM) or Egr-1 siRNA-1 (50 nM) transfected HMEC-1s (n = 3). (C, D) Confluent HMEC-1s transfected with scrambled siRNA (50 nM) or Egr-1 siRNA-1 (50 nM) were scratched and treated with SW480-derived EVs (1 µg/mL); then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). The number of migrated cells in the denuded zone of each group is shown in panel D. Scale bars represent 100 µm. Data are represented as mean ± SD. * P <0.05; ** P <0.01; *** P <0.001; n.s., not significant.

    Journal: PLoS ONE

    Article Title: Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

    doi: 10.1371/journal.pone.0115170

    Figure Lengend Snippet: (A) HMEC-1s were transfected with 50 nM of scrambled siRNA or Egr-1 siRNA-1, Egr-1 siRNA-2, or Egr-1 siRNA-3. mRNAs were isolated from the cells after 48 h and the level of Egr-1 mRNA was analyzed using real time RT-PCR (n = 3). (B) Nuclear translocation of Egr-1 protein after stimulation with SW480-derived EVs (1 µg/mL) for 1 h was analyzed in scrambled siRNA (50 nM) or Egr-1 siRNA-1 (50 nM) transfected HMEC-1s (n = 3). (C, D) Confluent HMEC-1s transfected with scrambled siRNA (50 nM) or Egr-1 siRNA-1 (50 nM) were scratched and treated with SW480-derived EVs (1 µg/mL); then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). The number of migrated cells in the denuded zone of each group is shown in panel D. Scale bars represent 100 µm. Data are represented as mean ± SD. * P <0.05; ** P <0.01; *** P <0.001; n.s., not significant.

    Article Snippet: Cells were treated with SW480-derived EVs (1 µg/mL, 0.5 mL), fixed with 4% paraformaldehyde, and incubated with rabbit anti-Egr-1 antibody (Cell Signaling Technology, Hitchin, United Kingdom) followed by AlexaFluor488 goat anti-rabbit IgG antibody (Invitrogen).

    Techniques: Transfection, Isolation, Quantitative RT-PCR, Translocation Assay, Derivative Assay

    (A, B) HMEC-1s were pretreated with signaling inhibitors for 1 h and then stimulated for 1 h with SW480-derived EVs (1 µg/mL). Nuclear translocation of Egr-1 protein was analyzed using confocal microscopy (n = 3). Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. Co-localized fluorescence signals (purple) indicate the translocation of Egr-1 into the nucleus. Representative photographs are shown in panel A. The percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus colocalized signals over total cells (B). (C, D) Confluent HMEC-1s were scratched and treated with SW480-derived EVs (1 µg/mL) in the presence or absence of signaling inhibitors; then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). Representative photographs of confocal microscopic imaging are shown in panel C and the number of migrated cells in the denuded zone of each group is shown in panel D. ERK1/2 inhibitor, PD98059 (20 µM); p38 MAPK inhibitor, SB203580 (10 µM); JNK inhibitor, SP600125 (20 µM); Akt inhibitor, BML-257 (20 µM). (E, F) C57BL/6 mice were subcutaneously injected with Matrigel containing SW480-derived EVs (20 µg) with PD98059 (20 µM) or SP600125 (20 µM). After 7 days, whole-mount staining of Matrigel with anti-CD31 antibody was conducted (n = 5). Representative confocal Z-stack photographs of whole mounts stained for CD31 (green) are shown in panel E. Fluorescence intensities of CD31 in the Z-stack plane of the Matrigel were measured as described in the (F). Scale bars in panels A, C, and E represent 30, 100, and 100 µm, respectively. Data are represented as mean ± SD. *** P <0.001.

    Journal: PLoS ONE

    Article Title: Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

    doi: 10.1371/journal.pone.0115170

    Figure Lengend Snippet: (A, B) HMEC-1s were pretreated with signaling inhibitors for 1 h and then stimulated for 1 h with SW480-derived EVs (1 µg/mL). Nuclear translocation of Egr-1 protein was analyzed using confocal microscopy (n = 3). Nuclei and Egr-1 proteins were stained with Hoechst (blue) and anti-Egr-1 antibody (red), respectively. Co-localized fluorescence signals (purple) indicate the translocation of Egr-1 into the nucleus. Representative photographs are shown in panel A. The percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus colocalized signals over total cells (B). (C, D) Confluent HMEC-1s were scratched and treated with SW480-derived EVs (1 µg/mL) in the presence or absence of signaling inhibitors; then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). Representative photographs of confocal microscopic imaging are shown in panel C and the number of migrated cells in the denuded zone of each group is shown in panel D. ERK1/2 inhibitor, PD98059 (20 µM); p38 MAPK inhibitor, SB203580 (10 µM); JNK inhibitor, SP600125 (20 µM); Akt inhibitor, BML-257 (20 µM). (E, F) C57BL/6 mice were subcutaneously injected with Matrigel containing SW480-derived EVs (20 µg) with PD98059 (20 µM) or SP600125 (20 µM). After 7 days, whole-mount staining of Matrigel with anti-CD31 antibody was conducted (n = 5). Representative confocal Z-stack photographs of whole mounts stained for CD31 (green) are shown in panel E. Fluorescence intensities of CD31 in the Z-stack plane of the Matrigel were measured as described in the (F). Scale bars in panels A, C, and E represent 30, 100, and 100 µm, respectively. Data are represented as mean ± SD. *** P <0.001.

    Article Snippet: Cells were treated with SW480-derived EVs (1 µg/mL, 0.5 mL), fixed with 4% paraformaldehyde, and incubated with rabbit anti-Egr-1 antibody (Cell Signaling Technology, Hitchin, United Kingdom) followed by AlexaFluor488 goat anti-rabbit IgG antibody (Invitrogen).

    Techniques: Derivative Assay, Translocation Assay, Confocal Microscopy, Staining, Fluorescence, Imaging, Injection

    (A) HMEC-1s were treated with DiI-labeled SW480-derived EVs (1 µg/mL) for 1 h in the presence or absence of MβCD (10 mM) (n = 3). SW480-derived EVs and nuclei were stained with DiI (red) and Hoechst (blue) respectively. Representative photographs are shown in panel A. Scale bars represent 10 µm. (B) Confluent HMEC-1s were scratched and treated with SW480-derived EVs (1 µg/mL) in the presence or absence of MβCD (10 mM); then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). (C, D) HMEC-1s were pretreated with MβCD (10 mM) for 1 h and then stimulated with SW480-derived EVs (1 µg/mL) for 1 h. Scale bars represent 30 µm. Nuclear translocation of Egr-1 protein was analyzed using confocal microscopy and the percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus co-localized signals over that of total cells (n = 3). Data are represented as mean ± SD. *** P <0.001.

    Journal: PLoS ONE

    Article Title: Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

    doi: 10.1371/journal.pone.0115170

    Figure Lengend Snippet: (A) HMEC-1s were treated with DiI-labeled SW480-derived EVs (1 µg/mL) for 1 h in the presence or absence of MβCD (10 mM) (n = 3). SW480-derived EVs and nuclei were stained with DiI (red) and Hoechst (blue) respectively. Representative photographs are shown in panel A. Scale bars represent 10 µm. (B) Confluent HMEC-1s were scratched and treated with SW480-derived EVs (1 µg/mL) in the presence or absence of MβCD (10 mM); then the number of migrated cells in the denuded zone was evaluated after 12 h (n = 3). (C, D) HMEC-1s were pretreated with MβCD (10 mM) for 1 h and then stimulated with SW480-derived EVs (1 µg/mL) for 1 h. Scale bars represent 30 µm. Nuclear translocation of Egr-1 protein was analyzed using confocal microscopy and the percentage of Egr-1-positive nuclei was determined by measuring the number of cells with nucleus co-localized signals over that of total cells (n = 3). Data are represented as mean ± SD. *** P <0.001.

    Article Snippet: Cells were treated with SW480-derived EVs (1 µg/mL, 0.5 mL), fixed with 4% paraformaldehyde, and incubated with rabbit anti-Egr-1 antibody (Cell Signaling Technology, Hitchin, United Kingdom) followed by AlexaFluor488 goat anti-rabbit IgG antibody (Invitrogen).

    Techniques: Labeling, Derivative Assay, Staining, Translocation Assay, Confocal Microscopy

    A. WT1 mRNA (+KTS and –KTS) splice variant PCR products expressed in LβT2 cells. Whole cell RNA was extracted from LβT2 cells, reverse transcribed to cDNA and quantified by real-time PCR, using specific primers to detect +KTS and –KTS splice variants, then displayed on a 1% agarose gel. Bands corresponding to the products for +KTS (301bp) and –KTS (292 bp) WT1 were detected in 4 independent samples of mRNA. B. WT1 and Egr1 protein expression in LβT2 cells. Cell proteins (30μg) were separated on 10% polyacrylamide-SDS gels, then analyzed by immunoblotting with specific antibodies for WT1, Egr1 and β-actin. Specific proteins were detected in the same samples of untreated LβT2 cells. C. Chromatin association of WT1 with the endogenous LHβ promoter in LβT2 cells. The association of WT1 and RNA Polymerase II with the LHβ promoter in untreated LβT2 cells was measured by Chromatin immunoprecipitation assays with antibodies against WT1 and phosphorylated RNApol II, as well as control (no Antibody). LHβ promoter occupancy was measured by quantitative real time PCR using primers specific for the LHβ promoter, and normalized for chromatin input in each sample. In this study, background binding (no Antibody) was set at 100% and association of RNA Polymerase II and WT1 are expressed relative to background values. Association was measured in 3 independent experiments with duplicate samples.

    Journal: PLoS ONE

    Article Title: A New Role for Wilms Tumor Protein 1: Differential Activities of + KTS and –KTS Variants to Regulate LHβ Transcription

    doi: 10.1371/journal.pone.0116825

    Figure Lengend Snippet: A. WT1 mRNA (+KTS and –KTS) splice variant PCR products expressed in LβT2 cells. Whole cell RNA was extracted from LβT2 cells, reverse transcribed to cDNA and quantified by real-time PCR, using specific primers to detect +KTS and –KTS splice variants, then displayed on a 1% agarose gel. Bands corresponding to the products for +KTS (301bp) and –KTS (292 bp) WT1 were detected in 4 independent samples of mRNA. B. WT1 and Egr1 protein expression in LβT2 cells. Cell proteins (30μg) were separated on 10% polyacrylamide-SDS gels, then analyzed by immunoblotting with specific antibodies for WT1, Egr1 and β-actin. Specific proteins were detected in the same samples of untreated LβT2 cells. C. Chromatin association of WT1 with the endogenous LHβ promoter in LβT2 cells. The association of WT1 and RNA Polymerase II with the LHβ promoter in untreated LβT2 cells was measured by Chromatin immunoprecipitation assays with antibodies against WT1 and phosphorylated RNApol II, as well as control (no Antibody). LHβ promoter occupancy was measured by quantitative real time PCR using primers specific for the LHβ promoter, and normalized for chromatin input in each sample. In this study, background binding (no Antibody) was set at 100% and association of RNA Polymerase II and WT1 are expressed relative to background values. Association was measured in 3 independent experiments with duplicate samples.

    Article Snippet: Membranes were then incubated with a WT1 primary antibody (C-19:SC-192 Santa Cruz Biotechnologies, Santa Cruz CA) overnight (1:500) or Egr1 primary antibody (Cell Signaling Technology) overnight (1:1000) at 4C followed by three 5 min washes with TBST and another incubation with secondary antibody, horseradish peroxidase-conjugated donkey anti-rabbit Fab fragment IgG (1:5000;GE HealthCare; Piscataway, New Jersey) for 2h.

    Techniques: Variant Assay, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Western Blot, Chromatin Immunoprecipitation, Binding Assay

    A. WT1 (-KTS) and (+ KTS) mRNA variant levels in LβT2 cells treated with 50nM GnRH for 90min. RNA was extracted and mRNAs levels measured by RT-PCR and normalized against GAPDH mRNA. Values for WT1 mRNAs are shown for both Reverse Transcriptase (RT) and no RT (negative control) conditions. Note that for no RT, the Y axis is interrupted and expanded to show the low values. Data is the mean ± SEM from 5–7 experiments. * = P<0.05, -GnRH vs +GnRH; ** = P<0.001, -GnRH vs +GnRH B. WT1 and Egr1 protein levels in LβT2 cells treated with 50nM GnRH for 0 to 3.5 h. The experiment was performed three times. Upper panel: Cells were lysed after GnRH treatment and proteins (30μg) were separated by 10% SDS-PAGE, then detected with antibodies against WT1 or Egr1. Immunoblotting for β-actin was performed on the same blots as WT1 and Egr1, and used for normalization of these proteins quantified by densitometry analysis. A representative blot is shown. Lower panel: Quantification of protein bands was performed with densitometry, and normalized protein levels are shown from combined experiments. Bands for Egr1 proteins were not detected (ND) in any blot at time zero without GnRH.

    Journal: PLoS ONE

    Article Title: A New Role for Wilms Tumor Protein 1: Differential Activities of + KTS and –KTS Variants to Regulate LHβ Transcription

    doi: 10.1371/journal.pone.0116825

    Figure Lengend Snippet: A. WT1 (-KTS) and (+ KTS) mRNA variant levels in LβT2 cells treated with 50nM GnRH for 90min. RNA was extracted and mRNAs levels measured by RT-PCR and normalized against GAPDH mRNA. Values for WT1 mRNAs are shown for both Reverse Transcriptase (RT) and no RT (negative control) conditions. Note that for no RT, the Y axis is interrupted and expanded to show the low values. Data is the mean ± SEM from 5–7 experiments. * = P<0.05, -GnRH vs +GnRH; ** = P<0.001, -GnRH vs +GnRH B. WT1 and Egr1 protein levels in LβT2 cells treated with 50nM GnRH for 0 to 3.5 h. The experiment was performed three times. Upper panel: Cells were lysed after GnRH treatment and proteins (30μg) were separated by 10% SDS-PAGE, then detected with antibodies against WT1 or Egr1. Immunoblotting for β-actin was performed on the same blots as WT1 and Egr1, and used for normalization of these proteins quantified by densitometry analysis. A representative blot is shown. Lower panel: Quantification of protein bands was performed with densitometry, and normalized protein levels are shown from combined experiments. Bands for Egr1 proteins were not detected (ND) in any blot at time zero without GnRH.

    Article Snippet: Membranes were then incubated with a WT1 primary antibody (C-19:SC-192 Santa Cruz Biotechnologies, Santa Cruz CA) overnight (1:500) or Egr1 primary antibody (Cell Signaling Technology) overnight (1:1000) at 4C followed by three 5 min washes with TBST and another incubation with secondary antibody, horseradish peroxidase-conjugated donkey anti-rabbit Fab fragment IgG (1:5000;GE HealthCare; Piscataway, New Jersey) for 2h.

    Techniques: Variant Assay, Reverse Transcription Polymerase Chain Reaction, Negative Control, SDS Page, Western Blot

    LβT2 cells were incubated with or without 50 nM GnRH and collected every 10 min for 120 min. ChIP assays were performed using antibody against WT1, Egr1 and phosphorylated pRNA Pol II. LHβ promoter occupancy was measured by quantitative real time PCR using primers for the LHβ promoter. The experiment was performed three times with duplicate samples and replicate PCR measurements in each sample. Data are presented as the mean + SEM and expressed as LHβ promoter occupancy relative to basal (no GnRH at time zero) binding.

    Journal: PLoS ONE

    Article Title: A New Role for Wilms Tumor Protein 1: Differential Activities of + KTS and –KTS Variants to Regulate LHβ Transcription

    doi: 10.1371/journal.pone.0116825

    Figure Lengend Snippet: LβT2 cells were incubated with or without 50 nM GnRH and collected every 10 min for 120 min. ChIP assays were performed using antibody against WT1, Egr1 and phosphorylated pRNA Pol II. LHβ promoter occupancy was measured by quantitative real time PCR using primers for the LHβ promoter. The experiment was performed three times with duplicate samples and replicate PCR measurements in each sample. Data are presented as the mean + SEM and expressed as LHβ promoter occupancy relative to basal (no GnRH at time zero) binding.

    Article Snippet: Membranes were then incubated with a WT1 primary antibody (C-19:SC-192 Santa Cruz Biotechnologies, Santa Cruz CA) overnight (1:500) or Egr1 primary antibody (Cell Signaling Technology) overnight (1:1000) at 4C followed by three 5 min washes with TBST and another incubation with secondary antibody, horseradish peroxidase-conjugated donkey anti-rabbit Fab fragment IgG (1:5000;GE HealthCare; Piscataway, New Jersey) for 2h.

    Techniques: Incubation, Real-time Polymerase Chain Reaction, Binding Assay

    LβT2 cells were transfected with siRNA against WT1 and a non-targeting siRNA as a control. After 72 h of incubation, cells were treated with or without GnRH for 1.5 h, followed by cell lysate collection, RNA extraction and western blot analysis (30 μg protein) on 10% PAGE-SDS gels. WT1 protein was detected by immunoblotting with specific antibodies for WT1, and normalized for β-actin on the same blot. A. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where the WT1 (-KTS) variant mRNA was reduced. B. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where both the WT1 (-KTS) and WT1 (+KTS) variant mRNAs were reduced. For each condition, the experiment was performed twice with 5 replicates. LHβ and Egr1 primary transcript mRNAs were normalized for GAPDH mRNA in the same sample. Control samples contained non-targeting siRNA. * p<.05 -GnRH vs +GnRH, in either Control siRNA or WT1 siRNA treatments. Values are mean ± SEM for 5 replicates. # = p<.05 -GnRH Control siRNA vs –GnRH WT1 siRNA, or p<.05+GnRH Control siRNA vs +GnRH WT1 siRNA.

    Journal: PLoS ONE

    Article Title: A New Role for Wilms Tumor Protein 1: Differential Activities of + KTS and –KTS Variants to Regulate LHβ Transcription

    doi: 10.1371/journal.pone.0116825

    Figure Lengend Snippet: LβT2 cells were transfected with siRNA against WT1 and a non-targeting siRNA as a control. After 72 h of incubation, cells were treated with or without GnRH for 1.5 h, followed by cell lysate collection, RNA extraction and western blot analysis (30 μg protein) on 10% PAGE-SDS gels. WT1 protein was detected by immunoblotting with specific antibodies for WT1, and normalized for β-actin on the same blot. A. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where the WT1 (-KTS) variant mRNA was reduced. B. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where both the WT1 (-KTS) and WT1 (+KTS) variant mRNAs were reduced. For each condition, the experiment was performed twice with 5 replicates. LHβ and Egr1 primary transcript mRNAs were normalized for GAPDH mRNA in the same sample. Control samples contained non-targeting siRNA. * p<.05 -GnRH vs +GnRH, in either Control siRNA or WT1 siRNA treatments. Values are mean ± SEM for 5 replicates. # = p<.05 -GnRH Control siRNA vs –GnRH WT1 siRNA, or p<.05+GnRH Control siRNA vs +GnRH WT1 siRNA.

    Article Snippet: Membranes were then incubated with a WT1 primary antibody (C-19:SC-192 Santa Cruz Biotechnologies, Santa Cruz CA) overnight (1:500) or Egr1 primary antibody (Cell Signaling Technology) overnight (1:1000) at 4C followed by three 5 min washes with TBST and another incubation with secondary antibody, horseradish peroxidase-conjugated donkey anti-rabbit Fab fragment IgG (1:5000;GE HealthCare; Piscataway, New Jersey) for 2h.

    Techniques: Transfection, Incubation, RNA Extraction, Western Blot, Expressing, Variant Assay

    WT1 (-KTS) may associate directly with the 3’Egr1 site of the LHβ promoter to stimulate basal promoter activity, but in the presence of GnRH stimulates Egr1 expression. Egr1 then binds to both the 3’- and 5’-Egr1 sites on the promoter and further increases LHβ transcription. WT1 (+KTS) may associate with the 3’-Egr1 site, but also requires the SF1 site for biological activity. Decreased expression of WT1 (+KTS) would stimulate basal LHβ expression as the suppressor is reduced. The isoforms likely compete for association to the LHβ promoter at the same sites.

    Journal: PLoS ONE

    Article Title: A New Role for Wilms Tumor Protein 1: Differential Activities of + KTS and –KTS Variants to Regulate LHβ Transcription

    doi: 10.1371/journal.pone.0116825

    Figure Lengend Snippet: WT1 (-KTS) may associate directly with the 3’Egr1 site of the LHβ promoter to stimulate basal promoter activity, but in the presence of GnRH stimulates Egr1 expression. Egr1 then binds to both the 3’- and 5’-Egr1 sites on the promoter and further increases LHβ transcription. WT1 (+KTS) may associate with the 3’-Egr1 site, but also requires the SF1 site for biological activity. Decreased expression of WT1 (+KTS) would stimulate basal LHβ expression as the suppressor is reduced. The isoforms likely compete for association to the LHβ promoter at the same sites.

    Article Snippet: Membranes were then incubated with a WT1 primary antibody (C-19:SC-192 Santa Cruz Biotechnologies, Santa Cruz CA) overnight (1:500) or Egr1 primary antibody (Cell Signaling Technology) overnight (1:1000) at 4C followed by three 5 min washes with TBST and another incubation with secondary antibody, horseradish peroxidase-conjugated donkey anti-rabbit Fab fragment IgG (1:5000;GE HealthCare; Piscataway, New Jersey) for 2h.

    Techniques: Activity Assay, Expressing

    Growth-quiescent SMCs were treated with MMP inhibitor GM6001+ (25 µM) and TAPI-1 (10 µM) for 30 min before stimulation with IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). ( A ) Egr-1 mRNA levels in GM6001+ or GM6001- treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( B ) Western blotting for Egr-1 or beta-actin in total extracts of SMCs treated with GM6001+ or GM6001-. ( C ) Egr-1 mRNA levels in TAPI-1 treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( D ) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with TAPI-1. DMSO was used as a carrier. ( E ) Western blotting for ADAM17, ADAM10, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ADAM17 siRNA (0.1 µM) and treated with IL-1beta for 30 min.

    Journal: PLoS ONE

    Article Title: IL-1beta Signals through the EGF Receptor and Activates Egr-1 through MMP-ADAM

    doi: 10.1371/journal.pone.0039811

    Figure Lengend Snippet: Growth-quiescent SMCs were treated with MMP inhibitor GM6001+ (25 µM) and TAPI-1 (10 µM) for 30 min before stimulation with IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). ( A ) Egr-1 mRNA levels in GM6001+ or GM6001- treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( B ) Western blotting for Egr-1 or beta-actin in total extracts of SMCs treated with GM6001+ or GM6001-. ( C ) Egr-1 mRNA levels in TAPI-1 treated SMCs treated with IL-1beta by qPCR. Data were normalized to beta-actin. ( D ) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with TAPI-1. DMSO was used as a carrier. ( E ) Western blotting for ADAM17, ADAM10, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ADAM17 siRNA (0.1 µM) and treated with IL-1beta for 30 min.

    Article Snippet: Rabbit monoclonal antibodies Egr-1 were obtained from Cell Signaling (Danvers, MA, USA).

    Techniques: Western Blot, Transfection

    Growth-quiescent SMCs were incubated with EGFR inhibitors AG1478 (5 µM) and PD153035 (5 µM) for 30 min, and exposed to IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). ( A ) Egr-1 mRNA levels in AG1478- and PD153035-treated SMCs incubated with IL-1beta by qPCR. Data were normalized to beta-actin. ( B ) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with AG1478 or PD153035. ( C ) Western blotting for EGFR, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with EGFR siRNA (0.1 µM) and treated with IL-1beta for 30 min. ( D ) Western blotting for ErbB4, EGFR, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ErbB4 siRNA (0.1 µM) and treated with IL-1beta for 30 min. ( E ) Egr-1 mRNA levels in AG825 (10 µM for 30 min)-treated SMCs incubated with IL-1beta for 30 min by qPCR. Data were normalized to beta-actin. ( F ) Western blotting for Egr-1 or beta-actin in total extracts of SMCs pretreated with AG825 (10 µM for 30 min) then stimulated with IL-1beta for another 30 min.

    Journal: PLoS ONE

    Article Title: IL-1beta Signals through the EGF Receptor and Activates Egr-1 through MMP-ADAM

    doi: 10.1371/journal.pone.0039811

    Figure Lengend Snippet: Growth-quiescent SMCs were incubated with EGFR inhibitors AG1478 (5 µM) and PD153035 (5 µM) for 30 min, and exposed to IL-1beta (10 ng/ml) for another 30 min (unless indicated otherwise). ( A ) Egr-1 mRNA levels in AG1478- and PD153035-treated SMCs incubated with IL-1beta by qPCR. Data were normalized to beta-actin. ( B ) Western blotting for Egr-1 or beta-actin of total extracts of SMCs treated with AG1478 or PD153035. ( C ) Western blotting for EGFR, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with EGFR siRNA (0.1 µM) and treated with IL-1beta for 30 min. ( D ) Western blotting for ErbB4, EGFR, Egr-1 or beta-actin of total extracts of SMCs transfected overnight with ErbB4 siRNA (0.1 µM) and treated with IL-1beta for 30 min. ( E ) Egr-1 mRNA levels in AG825 (10 µM for 30 min)-treated SMCs incubated with IL-1beta for 30 min by qPCR. Data were normalized to beta-actin. ( F ) Western blotting for Egr-1 or beta-actin in total extracts of SMCs pretreated with AG825 (10 µM for 30 min) then stimulated with IL-1beta for another 30 min.

    Article Snippet: Rabbit monoclonal antibodies Egr-1 were obtained from Cell Signaling (Danvers, MA, USA).

    Techniques: Incubation, Western Blot, Transfection

    ( A ) Growth-quiescent SMCs were stimulated with IL-1beta (10 ng/ml) for the indicated times. Total protein extracts were resolved by SDS-PAGE and subjected to Western blot analysis using antibodies for Egr-1, EGFR phospho-Tyr 845 , total EGFR and beta-actin. Alternatively, Western blotting for EGFR phospho-Tyr 845 or EGFR of total extracts of SMCs treated with ( B ) AG1478 (5 µM), ( C ) GM6001+ or GM6001- (25 µM) ( D ) TAPI-1 (10 µM), PD153035 (5 µM) for 30 min followed by IL-1beta stimulation for 5 min.

    Journal: PLoS ONE

    Article Title: IL-1beta Signals through the EGF Receptor and Activates Egr-1 through MMP-ADAM

    doi: 10.1371/journal.pone.0039811

    Figure Lengend Snippet: ( A ) Growth-quiescent SMCs were stimulated with IL-1beta (10 ng/ml) for the indicated times. Total protein extracts were resolved by SDS-PAGE and subjected to Western blot analysis using antibodies for Egr-1, EGFR phospho-Tyr 845 , total EGFR and beta-actin. Alternatively, Western blotting for EGFR phospho-Tyr 845 or EGFR of total extracts of SMCs treated with ( B ) AG1478 (5 µM), ( C ) GM6001+ or GM6001- (25 µM) ( D ) TAPI-1 (10 µM), PD153035 (5 µM) for 30 min followed by IL-1beta stimulation for 5 min.

    Article Snippet: Rabbit monoclonal antibodies Egr-1 were obtained from Cell Signaling (Danvers, MA, USA).

    Techniques: SDS Page, Western Blot

    ( A ) Western blotting for Egr-1, ADAM17, EGFR, IL-1RI or beta-actin using total extracts of growth-quiescent wild-type or ADAM17-deficient mEFs treated with IL-1beta for 30 or 60 min. ( B ) Interaction of 125 I-IL-1beta with ADAM17WT and ADAM17-deficient cells. Growth-quiescent ADAM17WT and ADAM17−/− mEFs (1.8×10 4 cells/well) were incubated with increasing amounts of 125 I-IL-1beta in 1% BSA/PBS for 1 h at 4°C. The cells were washed and lysed with 1M NaOH prior to assessment of counts in an automated gamma-counter.

    Journal: PLoS ONE

    Article Title: IL-1beta Signals through the EGF Receptor and Activates Egr-1 through MMP-ADAM

    doi: 10.1371/journal.pone.0039811

    Figure Lengend Snippet: ( A ) Western blotting for Egr-1, ADAM17, EGFR, IL-1RI or beta-actin using total extracts of growth-quiescent wild-type or ADAM17-deficient mEFs treated with IL-1beta for 30 or 60 min. ( B ) Interaction of 125 I-IL-1beta with ADAM17WT and ADAM17-deficient cells. Growth-quiescent ADAM17WT and ADAM17−/− mEFs (1.8×10 4 cells/well) were incubated with increasing amounts of 125 I-IL-1beta in 1% BSA/PBS for 1 h at 4°C. The cells were washed and lysed with 1M NaOH prior to assessment of counts in an automated gamma-counter.

    Article Snippet: Rabbit monoclonal antibodies Egr-1 were obtained from Cell Signaling (Danvers, MA, USA).

    Techniques: Western Blot, Incubation

    Oligonucleotides and plasmids.

    Journal: PLoS ONE

    Article Title: Transcriptional Regulation of Flotillins by the Extracellularly Regulated Kinases and Retinoid X Receptor Complexes

    doi: 10.1371/journal.pone.0045514

    Figure Lengend Snippet: Oligonucleotides and plasmids.

    Article Snippet: Rabbit monoclonal anti-Egr1 antibody was from Cell Signaling.

    Techniques: Luciferase, Plasmid Preparation, Expressing, Dominant Negative Mutation

    Flotillin promoter constructs F1-1330 (A, C, E) or F2-2130 (B, D, F) were cotransfected into Hela cells together with expression plasmids for Egr1, SRF, empty PSV control plasmid or with the ERK2 expression constructs, and the cells were lysed 48 h post-transfection. Relative luciferase activity of the control sample was set as 1. Values are mean ± standard deviation of at least 4 experiments measured in duplicates. ***p<0.001; **p<0.01; *p<0.05 vs. control.

    Journal: PLoS ONE

    Article Title: Transcriptional Regulation of Flotillins by the Extracellularly Regulated Kinases and Retinoid X Receptor Complexes

    doi: 10.1371/journal.pone.0045514

    Figure Lengend Snippet: Flotillin promoter constructs F1-1330 (A, C, E) or F2-2130 (B, D, F) were cotransfected into Hela cells together with expression plasmids for Egr1, SRF, empty PSV control plasmid or with the ERK2 expression constructs, and the cells were lysed 48 h post-transfection. Relative luciferase activity of the control sample was set as 1. Values are mean ± standard deviation of at least 4 experiments measured in duplicates. ***p<0.001; **p<0.01; *p<0.05 vs. control.

    Article Snippet: Rabbit monoclonal anti-Egr1 antibody was from Cell Signaling.

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

    Hela cells were transiently transfected with expression constructs for Egr1 or SRF. Empty PSV vector served as control. (A) Cell lysates were analyzed for flotillin-1 and -2 by Western blotting (left) and quantified (right). (B) RNA was isolated, transcribed into cDNA and analyzed by qPCR. Values are means ± standard deviation of 7 (Western Blot) or 4 (qPCR) experiments.*p<0.05, **, p<0.01 vs. control.

    Journal: PLoS ONE

    Article Title: Transcriptional Regulation of Flotillins by the Extracellularly Regulated Kinases and Retinoid X Receptor Complexes

    doi: 10.1371/journal.pone.0045514

    Figure Lengend Snippet: Hela cells were transiently transfected with expression constructs for Egr1 or SRF. Empty PSV vector served as control. (A) Cell lysates were analyzed for flotillin-1 and -2 by Western blotting (left) and quantified (right). (B) RNA was isolated, transcribed into cDNA and analyzed by qPCR. Values are means ± standard deviation of 7 (Western Blot) or 4 (qPCR) experiments.*p<0.05, **, p<0.01 vs. control.

    Article Snippet: Rabbit monoclonal anti-Egr1 antibody was from Cell Signaling.

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

    Chromatin immunoprecipitations were carried out in Hela cells as described in . DNA/protein complexes were precipitated with antibodies against RXR, RAR, Egr1 or HRS (IgG control). Coprecipitated DNA fragments were amplified with primers specific for approximately 500 bp (flotillin-1) (A) or 400 bp (flotillin-2) (B) of genomic DNA directly upstream of the ATG start codon. Results are representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: Transcriptional Regulation of Flotillins by the Extracellularly Regulated Kinases and Retinoid X Receptor Complexes

    doi: 10.1371/journal.pone.0045514

    Figure Lengend Snippet: Chromatin immunoprecipitations were carried out in Hela cells as described in . DNA/protein complexes were precipitated with antibodies against RXR, RAR, Egr1 or HRS (IgG control). Coprecipitated DNA fragments were amplified with primers specific for approximately 500 bp (flotillin-1) (A) or 400 bp (flotillin-2) (B) of genomic DNA directly upstream of the ATG start codon. Results are representative of two independent experiments.

    Article Snippet: Rabbit monoclonal anti-Egr1 antibody was from Cell Signaling.

    Techniques: Amplification

    A: mouse flotillin-2 proximal promoter sequence. The three putative binding sites for the RXR family and the two sites for Egr1 are shown in bold and underlined. The sequence shown in italics was deleted to remove the Egr1 binding sites. Mouse flotillin-2 promoter activity after PMA stimulation (B) or retinoic acid stimulation (C). Transfection and stimulation were performed as in .

    Journal: PLoS ONE

    Article Title: Transcriptional Regulation of Flotillins by the Extracellularly Regulated Kinases and Retinoid X Receptor Complexes

    doi: 10.1371/journal.pone.0045514

    Figure Lengend Snippet: A: mouse flotillin-2 proximal promoter sequence. The three putative binding sites for the RXR family and the two sites for Egr1 are shown in bold and underlined. The sequence shown in italics was deleted to remove the Egr1 binding sites. Mouse flotillin-2 promoter activity after PMA stimulation (B) or retinoic acid stimulation (C). Transfection and stimulation were performed as in .

    Article Snippet: Rabbit monoclonal anti-Egr1 antibody was from Cell Signaling.

    Techniques: Sequencing, Binding Assay, Activity Assay, Transfection

    ( A ) IVT-synthesized Egr-1 binds to ORF50 P probes. EMSA studies were performed using IVT-synthesized Egr-1 products and DIG-labeled ORF50 P probes (see ). ( B ) Mutations in the putative Egr-1 binding domain inhibit Egr-1 binding. EMSA experiments were performed using wt ORF50 P3 and ORF50 P8 probes as well as corresponding probes displaying mutations in the suspected Egr-1 binding domain ( ORF50 P3m and ORF50 P8m). ( C ) Nuclear lysates from KSHV-infected cells formed a complex with ORF50 P probes. BCBL-1 cells were synchronized in S phase of cell cycle according to earlier protocols , treated with 20 ng/ml TPA for 8 h, and lysed. Nuclear extracts containing Egr-1 proteins were used to perform EMSA studies using ORF50 P3 and ORF50 P8 probes. Specific Egr-1 binding was confirmed by performing supershifts using specific antibodies to Egr-1 ( lanes 4 and 8 ) or nonspecific IgGs ( lanes 3 and 7 ). The arrowhead indicates protein/DNA complex formation. Specific antibody/protein/DNA supershifts are denoted by the asterisk.

    Journal: PLoS ONE

    Article Title: Resveratrol Inhibits KSHV Reactivation by Lowering the Levels of Cellular EGR-1

    doi: 10.1371/journal.pone.0033364

    Figure Lengend Snippet: ( A ) IVT-synthesized Egr-1 binds to ORF50 P probes. EMSA studies were performed using IVT-synthesized Egr-1 products and DIG-labeled ORF50 P probes (see ). ( B ) Mutations in the putative Egr-1 binding domain inhibit Egr-1 binding. EMSA experiments were performed using wt ORF50 P3 and ORF50 P8 probes as well as corresponding probes displaying mutations in the suspected Egr-1 binding domain ( ORF50 P3m and ORF50 P8m). ( C ) Nuclear lysates from KSHV-infected cells formed a complex with ORF50 P probes. BCBL-1 cells were synchronized in S phase of cell cycle according to earlier protocols , treated with 20 ng/ml TPA for 8 h, and lysed. Nuclear extracts containing Egr-1 proteins were used to perform EMSA studies using ORF50 P3 and ORF50 P8 probes. Specific Egr-1 binding was confirmed by performing supershifts using specific antibodies to Egr-1 ( lanes 4 and 8 ) or nonspecific IgGs ( lanes 3 and 7 ). The arrowhead indicates protein/DNA complex formation. Specific antibody/protein/DNA supershifts are denoted by the asterisk.

    Article Snippet: Rabbit antibodies to phospho-ERK1/2, total ERK1/2, actin, and Egr-1 (15F7; monoclonal antibodies) purchased from Cell Signaling Technology, Beverly, MA were used in this study.

    Techniques: Synthesized, Labeling, Binding Assay, Infection

    ( A ) BCBL-1 cells were synchronized in S phase and treated with 20 ng/ml of TPA for 8 h. ChIP assays were performed using 2 µg of specific antibodies to Egr-1 or nonspecific IgGs. Primers specifically targeting ORF50 P3 or ORF50 P8 (see ) were used to perform semi-quantitative PCR experiments on 1% of total DNA (input) and IP samples. Respective cDNA at 10, 15, 20, 25, and 30 cycles were removed and resolved on a 2% agarose gel. IP of BCBL-1 DNA using specific antibodies to histone H3 was used as positive controls. ( B ) A schematic representation of ORF50P used to make the deletions of the luciferase reporter constructs. The nucleotide locations correspond to the old KSHV genome sequence NC_003409 which has since been updated to NC_009333.1. Asterisks refer to the ORF50P3 and ORF50P8 locations, respectively. ( C ) Overexpression of Egr-1 activates ORF50P via interacting with ORF50P3 and ORF50P8 domains. HEK293 cells were co-transfected with a combination of three vectors, one from the following groups: (i) pcDNA3.1(+) or egr-1 /pcDNA3.1(+), (ii) the control vector, pRL-TK, and (iii) empty pGL3 vectors or pGL3 vectors encoding FL ORF50P or one of several deletions (Δ-2922 to -2044; Δ-2922 to -1322; Δ-2922 to -894; and Δ-2922 to -169). After 48 h post-transfection, the cells were lysed, and relative luciferase activity was monitored. Firefly luciferase was normalized to the corresponding Renilla luciferase activity. The luciferase activation of pGL3 by egr-1 /pcDNA3.1(+) was represented as 1-fold. Each point denotes the average ± SD of three experiments. Columns with different alphabets are statistically significant (P<0.05) by least significant difference (LSD).

    Journal: PLoS ONE

    Article Title: Resveratrol Inhibits KSHV Reactivation by Lowering the Levels of Cellular EGR-1

    doi: 10.1371/journal.pone.0033364

    Figure Lengend Snippet: ( A ) BCBL-1 cells were synchronized in S phase and treated with 20 ng/ml of TPA for 8 h. ChIP assays were performed using 2 µg of specific antibodies to Egr-1 or nonspecific IgGs. Primers specifically targeting ORF50 P3 or ORF50 P8 (see ) were used to perform semi-quantitative PCR experiments on 1% of total DNA (input) and IP samples. Respective cDNA at 10, 15, 20, 25, and 30 cycles were removed and resolved on a 2% agarose gel. IP of BCBL-1 DNA using specific antibodies to histone H3 was used as positive controls. ( B ) A schematic representation of ORF50P used to make the deletions of the luciferase reporter constructs. The nucleotide locations correspond to the old KSHV genome sequence NC_003409 which has since been updated to NC_009333.1. Asterisks refer to the ORF50P3 and ORF50P8 locations, respectively. ( C ) Overexpression of Egr-1 activates ORF50P via interacting with ORF50P3 and ORF50P8 domains. HEK293 cells were co-transfected with a combination of three vectors, one from the following groups: (i) pcDNA3.1(+) or egr-1 /pcDNA3.1(+), (ii) the control vector, pRL-TK, and (iii) empty pGL3 vectors or pGL3 vectors encoding FL ORF50P or one of several deletions (Δ-2922 to -2044; Δ-2922 to -1322; Δ-2922 to -894; and Δ-2922 to -169). After 48 h post-transfection, the cells were lysed, and relative luciferase activity was monitored. Firefly luciferase was normalized to the corresponding Renilla luciferase activity. The luciferase activation of pGL3 by egr-1 /pcDNA3.1(+) was represented as 1-fold. Each point denotes the average ± SD of three experiments. Columns with different alphabets are statistically significant (P<0.05) by least significant difference (LSD).

    Article Snippet: Rabbit antibodies to phospho-ERK1/2, total ERK1/2, actin, and Egr-1 (15F7; monoclonal antibodies) purchased from Cell Signaling Technology, Beverly, MA were used in this study.

    Techniques: Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Luciferase, Construct, Sequencing, Over Expression, Transfection, Plasmid Preparation, Activity Assay, Activation Assay

    HEK293 cells were infected with KSHV at 5 MOI, incubated at 37°C at different time points up to 48 hPI, and lysed. ( A ) Expression of KSHV RTA and Egr-1 were elevated up to 6 hPI. SDS-PAGE was performed using uninfected ( lane 1 ) or KSHV-infected cell lysates ( lanes 2–7 ). The samples were run on a 10% gel and transferred to a PVDF membrane. Western blotting was performed using specific Egr-1 or KSHV RTA Abs. Abs targeting β-actin were used as internal controls. ( B ) Analysis of ORF50 and egr-1 transcription. KSHV-infected HEK293 cells were lysed and RNA was extracted. Next, cDNA was synthesized and subjected to quantitative real-time PCR analysis. Baseline expression of genes at 1 hPI was considered as 1-fold for comparisons. Each point denotes the average±S.D. ( error bars ) of three experiments.

    Journal: PLoS ONE

    Article Title: Resveratrol Inhibits KSHV Reactivation by Lowering the Levels of Cellular EGR-1

    doi: 10.1371/journal.pone.0033364

    Figure Lengend Snippet: HEK293 cells were infected with KSHV at 5 MOI, incubated at 37°C at different time points up to 48 hPI, and lysed. ( A ) Expression of KSHV RTA and Egr-1 were elevated up to 6 hPI. SDS-PAGE was performed using uninfected ( lane 1 ) or KSHV-infected cell lysates ( lanes 2–7 ). The samples were run on a 10% gel and transferred to a PVDF membrane. Western blotting was performed using specific Egr-1 or KSHV RTA Abs. Abs targeting β-actin were used as internal controls. ( B ) Analysis of ORF50 and egr-1 transcription. KSHV-infected HEK293 cells were lysed and RNA was extracted. Next, cDNA was synthesized and subjected to quantitative real-time PCR analysis. Baseline expression of genes at 1 hPI was considered as 1-fold for comparisons. Each point denotes the average±S.D. ( error bars ) of three experiments.

    Article Snippet: Rabbit antibodies to phospho-ERK1/2, total ERK1/2, actin, and Egr-1 (15F7; monoclonal antibodies) purchased from Cell Signaling Technology, Beverly, MA were used in this study.

    Techniques: Infection, Incubation, Expressing, SDS Page, Western Blot, Synthesized, Real-time Polymerase Chain Reaction

    ( A ) Elevated Egr-1 was observed in cells transfected with egr-1/pCDNA3.1(+). BCBL-1 cells were untransfected, mock transfected, transfected with pcDNA3.1 or egr-1 /pcDNA3.1. The cells to be used as controls ( lanes 1–3 ) were incubated at 37°C for 48 h while the cells transfected with egr-1 /pcDNA3.1 were incubated for 24, 48, and 72 h, respectively. At the end of incubation, the cells were lysed and the lysates were used to perform Western blotting. ( B ) Effect of elevated Egr-1 on KSHV ORF50 and ORF8 expression. BCBL-1 cells were untransfected or transfected as described above. RNA was extracted, cDNA was synthesized, and the expression of cellular egr-1 ; and KSHV encoded ORF50 and ORF8 were analyzed by qRT-PCR. Baseline expression of genes was considered as 1-fold for comparisons. Each point denotes the average±S.D. ( error bars ) of three experiments. ( C ) Expression of lytic proteins in BCBL-1 cells transfected with Egr-1. KSHV-infected cells were untransfected, transfected with pcDNA3.1, or egr-1 /pcDNA3.1 for 48 h. The stained cells examined using a confocal microscope (magnification ×62). The average number of fluorescent cells were counted over five random fields and used for comparisons. ( D ) Enhanced egr-1 activates MAPK signaling in BCBL-1 cells. KSHV-infected cells were untransfected, mock-transfected, transfected with pcDNA3.1(+), or egr-1 /pcDNA3.1(+) for 48 h. Each group of cells was left untreated or they were treated with 10 µM of U0126 1 h prior to transfection and remained throughout the incubation period. Cell lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. ( E ) U0126 inhibits phosho-ERK1/2 and Egr-1. Briefly, BCBL-1 cells were treated with different concentrations of U0126. Following 24 h incubation, the cells were lysed and the lysates were used to perform Western blotting as per earlier protocols using specific antibodies.

    Journal: PLoS ONE

    Article Title: Resveratrol Inhibits KSHV Reactivation by Lowering the Levels of Cellular EGR-1

    doi: 10.1371/journal.pone.0033364

    Figure Lengend Snippet: ( A ) Elevated Egr-1 was observed in cells transfected with egr-1/pCDNA3.1(+). BCBL-1 cells were untransfected, mock transfected, transfected with pcDNA3.1 or egr-1 /pcDNA3.1. The cells to be used as controls ( lanes 1–3 ) were incubated at 37°C for 48 h while the cells transfected with egr-1 /pcDNA3.1 were incubated for 24, 48, and 72 h, respectively. At the end of incubation, the cells were lysed and the lysates were used to perform Western blotting. ( B ) Effect of elevated Egr-1 on KSHV ORF50 and ORF8 expression. BCBL-1 cells were untransfected or transfected as described above. RNA was extracted, cDNA was synthesized, and the expression of cellular egr-1 ; and KSHV encoded ORF50 and ORF8 were analyzed by qRT-PCR. Baseline expression of genes was considered as 1-fold for comparisons. Each point denotes the average±S.D. ( error bars ) of three experiments. ( C ) Expression of lytic proteins in BCBL-1 cells transfected with Egr-1. KSHV-infected cells were untransfected, transfected with pcDNA3.1, or egr-1 /pcDNA3.1 for 48 h. The stained cells examined using a confocal microscope (magnification ×62). The average number of fluorescent cells were counted over five random fields and used for comparisons. ( D ) Enhanced egr-1 activates MAPK signaling in BCBL-1 cells. KSHV-infected cells were untransfected, mock-transfected, transfected with pcDNA3.1(+), or egr-1 /pcDNA3.1(+) for 48 h. Each group of cells was left untreated or they were treated with 10 µM of U0126 1 h prior to transfection and remained throughout the incubation period. Cell lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. ( E ) U0126 inhibits phosho-ERK1/2 and Egr-1. Briefly, BCBL-1 cells were treated with different concentrations of U0126. Following 24 h incubation, the cells were lysed and the lysates were used to perform Western blotting as per earlier protocols using specific antibodies.

    Article Snippet: Rabbit antibodies to phospho-ERK1/2, total ERK1/2, actin, and Egr-1 (15F7; monoclonal antibodies) purchased from Cell Signaling Technology, Beverly, MA were used in this study.

    Techniques: Transfection, Incubation, Western Blot, Expressing, Synthesized, Quantitative RT-PCR, Infection, Staining, Microscopy, SDS Page

    ( A ) Resveratrol inhibited egr-1 expression by as early as 6 hPI in HEK293 cells. HEK293 cells were infected with 5 MOI of KSHV for 2 h at 37°C. After infection, the cells were washed and cultured in growth medium in the presence or absence of different concentrations of resveratrol for 4 h. At the end of incubation, the cells were lysed, RNA was extracted, cDNA was synthesized, and egr-1 levels were analyzed by qRT-PCR. Each point denotes the average±S.D. ( error bars ) of three experiments. ( B ) Resveratrol inhibits MAPK activity during de novo KSHV infection. HEK293 cells were mock-infected or infected with 5 MOI of KSHV for 2 h at 37°C. After infection, the cells were washed and cultured in growth medium in the presence or absence of 100 µM of resveratrol for 48 h. The cells were lysed using gold lysis buffer (GLB) and the lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies.

    Journal: PLoS ONE

    Article Title: Resveratrol Inhibits KSHV Reactivation by Lowering the Levels of Cellular EGR-1

    doi: 10.1371/journal.pone.0033364

    Figure Lengend Snippet: ( A ) Resveratrol inhibited egr-1 expression by as early as 6 hPI in HEK293 cells. HEK293 cells were infected with 5 MOI of KSHV for 2 h at 37°C. After infection, the cells were washed and cultured in growth medium in the presence or absence of different concentrations of resveratrol for 4 h. At the end of incubation, the cells were lysed, RNA was extracted, cDNA was synthesized, and egr-1 levels were analyzed by qRT-PCR. Each point denotes the average±S.D. ( error bars ) of three experiments. ( B ) Resveratrol inhibits MAPK activity during de novo KSHV infection. HEK293 cells were mock-infected or infected with 5 MOI of KSHV for 2 h at 37°C. After infection, the cells were washed and cultured in growth medium in the presence or absence of 100 µM of resveratrol for 48 h. The cells were lysed using gold lysis buffer (GLB) and the lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies.

    Article Snippet: Rabbit antibodies to phospho-ERK1/2, total ERK1/2, actin, and Egr-1 (15F7; monoclonal antibodies) purchased from Cell Signaling Technology, Beverly, MA were used in this study.

    Techniques: Expressing, Infection, Cell Culture, Incubation, Synthesized, Quantitative RT-PCR, Activity Assay, Lysis, SDS Page, Western Blot

    ( A ) RDS lowers Egr-1 and KSHV RTA expression. KSHV-infected BCBL-1 cells were synchronized in S phase and untreated or treated using 20 ng/ml of TPA for 2 h. Each group of cells was left untreated or was further treated using filtered or unfiltered RDS containing 100 µM of resveratrol. The cells were incubated at 37°C for 6 h and lysed. The lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. ( B ) RDS reduces the number of KSHV-infected cells undergoing reactivation in HEK293 cells. Mock-infected, KSHV-infected, and KSHV-infected cells in the presence of RDS containing 100 µM resveratrol were stained using monoclonal mouse anti-Egr-1 IgGs and rabbit peptide antibodies targeting KSHV RTA and examined under a fluorescent microscope (magnification ×100). ( C ) Overexpression of Egr-1 is unable to overcome RDS-mediated inhibition of virus reactivation. BCBL-1 cells were transiently transfected using pcDNA3.1(+) or egr-1 /pcDNA3.1(+) and subsequently treated with unfiltered RDS containing 100 µM of Resveratrol for 6 h. RNA was extracted, cDNA was synthesized, and egr-1 and KSHV ORF50 were analyzed by qRT-PCR. Each point denotes the average±S.D. ( error bars ) of three experiments. ( D ) RDS lowers egr-1 and KSHV ORF50 transcriptional activity. BCBL-1 cells were synchronized in S phase, treated with 20 ng/ml of TPA, and treated using filtered or unfiltered RDS containing 100 µM of resveratrol as before. RNA was extracted, cDNA was synthesized, and egr-1 and KSHV ORF50 were analyzed by qRT-PCR. Each point denotes the average±S.D. ( error bars ) of three experiments. Columns with different alphabets are statistically significant (P<0.05) by LSD. ( E ) RDS inhibits the ability of Egr-1 to bind KSHV ORF50 in vivo . BCBL-1 cells were synchronized and treated with TPA as before. The cells were further treated using unfiltered RDS containing 100 µM of resveratrol and incubated for 6 h. ChIP assays were performed using 2 µg of specific Egr-1 Abs. Semi-quantitative PCR experiments were performed using samples from 1% input DNA and IP samples in order to determine the expression of ORF50 P8. Respective cDNA at 10, 15, 20, 25, and 30 cycles were resolved on a 2% agarose gel. Specific antibodies to histone H3 and nonspecific IgGs were used to IP sample chromatin and served as positive and negative controls, respectively.

    Journal: PLoS ONE

    Article Title: Resveratrol Inhibits KSHV Reactivation by Lowering the Levels of Cellular EGR-1

    doi: 10.1371/journal.pone.0033364

    Figure Lengend Snippet: ( A ) RDS lowers Egr-1 and KSHV RTA expression. KSHV-infected BCBL-1 cells were synchronized in S phase and untreated or treated using 20 ng/ml of TPA for 2 h. Each group of cells was left untreated or was further treated using filtered or unfiltered RDS containing 100 µM of resveratrol. The cells were incubated at 37°C for 6 h and lysed. The lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. ( B ) RDS reduces the number of KSHV-infected cells undergoing reactivation in HEK293 cells. Mock-infected, KSHV-infected, and KSHV-infected cells in the presence of RDS containing 100 µM resveratrol were stained using monoclonal mouse anti-Egr-1 IgGs and rabbit peptide antibodies targeting KSHV RTA and examined under a fluorescent microscope (magnification ×100). ( C ) Overexpression of Egr-1 is unable to overcome RDS-mediated inhibition of virus reactivation. BCBL-1 cells were transiently transfected using pcDNA3.1(+) or egr-1 /pcDNA3.1(+) and subsequently treated with unfiltered RDS containing 100 µM of Resveratrol for 6 h. RNA was extracted, cDNA was synthesized, and egr-1 and KSHV ORF50 were analyzed by qRT-PCR. Each point denotes the average±S.D. ( error bars ) of three experiments. ( D ) RDS lowers egr-1 and KSHV ORF50 transcriptional activity. BCBL-1 cells were synchronized in S phase, treated with 20 ng/ml of TPA, and treated using filtered or unfiltered RDS containing 100 µM of resveratrol as before. RNA was extracted, cDNA was synthesized, and egr-1 and KSHV ORF50 were analyzed by qRT-PCR. Each point denotes the average±S.D. ( error bars ) of three experiments. Columns with different alphabets are statistically significant (P<0.05) by LSD. ( E ) RDS inhibits the ability of Egr-1 to bind KSHV ORF50 in vivo . BCBL-1 cells were synchronized and treated with TPA as before. The cells were further treated using unfiltered RDS containing 100 µM of resveratrol and incubated for 6 h. ChIP assays were performed using 2 µg of specific Egr-1 Abs. Semi-quantitative PCR experiments were performed using samples from 1% input DNA and IP samples in order to determine the expression of ORF50 P8. Respective cDNA at 10, 15, 20, 25, and 30 cycles were resolved on a 2% agarose gel. Specific antibodies to histone H3 and nonspecific IgGs were used to IP sample chromatin and served as positive and negative controls, respectively.

    Article Snippet: Rabbit antibodies to phospho-ERK1/2, total ERK1/2, actin, and Egr-1 (15F7; monoclonal antibodies) purchased from Cell Signaling Technology, Beverly, MA were used in this study.

    Techniques: Expressing, Infection, Incubation, SDS Page, Western Blot, Staining, Microscopy, Over Expression, Inhibition, Transfection, Synthesized, Quantitative RT-PCR, Activity Assay, In Vivo, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis