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  • 99
    Thermo Fisher gene exp gapdh mm99999915 g1
    TRBP Phosphorylation Reduces Merlin Binding and TRBP Polyubiquitination (A) Representative immunoblot (left) and HMW TRBP protein levels, quantified from immunoblot of lysates from hippocampal neurons treated with vehicle (DMSO) or MG132 (60 min), normalized to <t>GAPDH</t> and plotted relative to vehicle alone (set as 1.0). (B) Representative immunoblot (left) and quantification of TRBP protein bound to GST-S5a (ubiquitin binding motif) (right) using lysates from hippocampal neurons pre-treated with either U0126 or vehicle control (30 min), followed by BDNF (60 min). Ubiquitinated TRBP protein was normalized to total ubiquitin pull-down and plotted relative to vehicle (set as 1.0). *non-TRBP specific band reactive with secondary antibody. (C) Lysates from HEK293T cells co-expressing FL-TRBPWT, SΔA, SΔD, or PCDNA3.1 alone (control) with HA-K48 ubiquitin underwent stringent IP with anti-FLAG antibody, followed by immunoblot with anti-FLAG and anti-HA antibodies. Shown are representative immunoblots (left) and quantification (right) of co-associated HA-K48 ubiquitin normalized to the amount of immunoprecipitated FL-TRBP for each construct, plotted relative to the FL-TRBPWT condition (set as 1.0). (D) Lysates from HEK293T cells co-expressing FL-TRBPWT, SΔA, SΔD, or PCDNA3.1 alone (control) with HA-Merlin were immunoprecipitated with anti-FLAG antibody, followed by immunoblot with anti-FLAG and anti-HA antibodies. Shown are representative immunoblots (left) and quantification (right) of co-associated HA-Merlin protein normalized first to the amount of input HA-Merlin protein and next to the immunoprecipitated FL-TRBP for each construct, plotted relative to the FL-TRBPWT condition (set as 1.0). .
    Gene Exp Gapdh Mm99999915 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8768 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gapdh
    Western blot analysis of transforming growth factor beta <t>(TGF‐β)</t> and alpha‐smooth muscle actin (α‐SMA). (a) Cytoplasmic fractions were analyzed by WB with TGF‐β and α‐SMA and glyceraldehyde phosphate dehydrogenase <t>(GAPDH)</t> antibodies. (b) Arbitrary values expressed as mean and SD. * P
    Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16774 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gapdh
    Western blot analysis of transforming growth factor beta <t>(TGF‐β)</t> and alpha‐smooth muscle actin (α‐SMA). (a) Cytoplasmic fractions were analyzed by WB with TGF‐β and α‐SMA and glyceraldehyde phosphate dehydrogenase <t>(GAPDH)</t> antibodies. (b) Arbitrary values expressed as mean and SD. * P
    Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 30834 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology gapdh
    Mapping ELK1 polypeptide segments required for co-activation by AR(A/B) by internal deletion analysis. A shows data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream <t>Gal4</t> elements ( GAL4-TATA-LUC ) and also with a vector expressing the AR A/B domain fused to the VP16 transactivation domain. Cells were transfected with plasmids expressing Gal4 fusion proteins of ELK1. The fusion constructs substituted the Gal4 DNA binding domain ( Gal4-DBD ) for the ETS DNA binding domain of ELK1. Within this fusion construct, a series of internal deletions were made, as indicated in the schematic in A . Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The promoter activity shown on the y axis required the presence of the AR A/B domain because knocking down AR(A/B) expression in the same cells transfected with full-length Gal4-ELK1 decreased the promoter activity to the basal value shown in the figure for Gal4-DBD alone. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or <t>GAPDH</t> (loading control). B shows data obtained using recombinant HeLa cells generated by stably transducing only GAL4-TATA-LUC . The cells were transfected with each of the Gal4-ELK1 fusion constructs used in A and co-transfected with an expression plasmid for a constitutively active mutant of MEK1 or with the vector control. Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). C shows a schematic of the domain organization of ELK1; here, the deletion mapping of two ELK1 polypeptide segments encompassing residues required for association with AR(A/B) (data from Figs. 3 A , 4 A, and 5 A ) is represented by gray shading of the two segments. For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. In all panels, the error bars represent standard deviation of experimental triplicates. *, p
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    gapdh  (Abcam)
    99
    Abcam gapdh
    Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of IκBα and p-IκBα protein; (D-F) Expression of nuclear and cytosolic p65 protein. Histone <t>H3</t> served as the nuclear loading control, <t>GAPDH</t> served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P
    Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 18714 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti gapdh
    <t>PKCε</t> protein levels in HeLaPKCεA/E and glioblastoma cell lines. Protein extracts from HeLaPKCεA/E (−Dox), HeLaPKCεA/E (+Dox), U-118 MG, U-138 MG, T98G, LN18 cell lines were subjected to Western blot analysis using antibodies against PKCε and <t>GAPDH.</t> HeLaPKCεA/E cells with doxycycline-induced expression of PKCε (+Dox) and without doxycycline treatment (−Dox) served as a reference cell lines with low and high expression of PKCε, respectively. The average relative absorbance of three independent experiments is presented in a bar graph
    Anti Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 12074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti gapdh
    <t>PKCε</t> protein levels in HeLaPKCεA/E and glioblastoma cell lines. Protein extracts from HeLaPKCεA/E (−Dox), HeLaPKCεA/E (+Dox), U-118 MG, U-138 MG, T98G, LN18 cell lines were subjected to Western blot analysis using antibodies against PKCε and <t>GAPDH.</t> HeLaPKCεA/E cells with doxycycline-induced expression of PKCε (+Dox) and without doxycycline treatment (−Dox) served as a reference cell lines with low and high expression of PKCε, respectively. The average relative absorbance of three independent experiments is presented in a bar graph
    Anti Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 10766 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp gapdh hs99999905 m1
    <t>PKCε</t> protein levels in HeLaPKCεA/E and glioblastoma cell lines. Protein extracts from HeLaPKCεA/E (−Dox), HeLaPKCεA/E (+Dox), U-118 MG, U-138 MG, T98G, LN18 cell lines were subjected to Western blot analysis using antibodies against PKCε and <t>GAPDH.</t> HeLaPKCεA/E cells with doxycycline-induced expression of PKCε (+Dox) and without doxycycline treatment (−Dox) served as a reference cell lines with low and high expression of PKCε, respectively. The average relative absorbance of three independent experiments is presented in a bar graph
    Gene Exp Gapdh Hs99999905 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gapdh
    <t>PKCε</t> protein levels in HeLaPKCεA/E and glioblastoma cell lines. Protein extracts from HeLaPKCεA/E (−Dox), HeLaPKCεA/E (+Dox), U-118 MG, U-138 MG, T98G, LN18 cell lines were subjected to Western blot analysis using antibodies against PKCε and <t>GAPDH.</t> HeLaPKCεA/E cells with doxycycline-induced expression of PKCε (+Dox) and without doxycycline treatment (−Dox) served as a reference cell lines with low and high expression of PKCε, respectively. The average relative absorbance of three independent experiments is presented in a bar graph
    Gapdh, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14534 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti gapdh
    <t>PKCε</t> protein levels in HeLaPKCεA/E and glioblastoma cell lines. Protein extracts from HeLaPKCεA/E (−Dox), HeLaPKCεA/E (+Dox), U-118 MG, U-138 MG, T98G, LN18 cell lines were subjected to Western blot analysis using antibodies against PKCε and <t>GAPDH.</t> HeLaPKCεA/E cells with doxycycline-induced expression of PKCε (+Dox) and without doxycycline treatment (−Dox) served as a reference cell lines with low and high expression of PKCε, respectively. The average relative absorbance of three independent experiments is presented in a bar graph
    Anti Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7859 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti gapdh
    TCR signaling induces RORγt phosphorylation and subsequent <t>AhR-RORγt</t> interaction. ( A ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, p-IKKβ (Ser 180/181 ), and IKKβ in primary splenic T cells. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( B ) Coimmunoprecipitation of endogenous AhR with RORγt from lysates of murine primary splenic T cells stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( C ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, and IKKβ in primary splenic T cells of IKKβ f/f or CD4-Cre;IKKβ f/f mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( D ) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt (left) or between AhR and Ser 489 -phosphorylated RORγt (right) in primary T cells of IKKβ f/f or IKKβ f/f ;CD4-Cre mice. T cells were stimulated as in (C). Each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Original magnification, ×630; scale bars, 10 μm. ( E and F ) ELISA of various cytokines in supernatants of primary splenic T cells from IKKβ f/f or IKKβ f/f ;CD4-Cre mice (E), as well as RORγt f/f or RORγt f/f ;CD4-Cre mice (F). T cells were stimulated with plate-bound anti-CD3 antibodies (2 μg each per milliliter) for 3 days. Means ± SD are shown. n = 3 per group. ( G ) Immunoblotting of RORγt and <t>GAPDH</t> proteins from primary splenic T cells of RORγt f/f or RORγt f/f ;CD4-Cre mice. Data shown (A to G) are representative of three independent experiments. ( H ) Schematic model of IL-17A transcription induced by the AhR-RORγt complex in GLK-overexpressing or TCR-stimulated T cells. GLK overexpression in T cells of T cell–specific GLK Tg (Lck-GLK Tg) mice induces AhR Ser 36 phosphorylation through PKCθ and also induces RORγt Ser 489 phosphorylation through IKKβ. Once RORγt is phosphorylated, RORγt interacts directly with AhR. Phosphorylated AhR is responsible for transporting RORγt into cell nucleus. The AhR-RORγt complex binds to both the RORγt-binding element (−877 to −872) and the AhR-binding element (−254 to −249) of the IL-17A promoter, leading to induction of IL-17A transcription. In normal T cells, TCR stimulation also induces GLK kinase activity and downstream signaling, including IKKβ activation, RORγt Ser 489 phosphorylation, and the AhR-RORγt interaction. Besides NF-κB, other critical transcription factors [such as nuclear factor of activated T cell 1 (NFAT1) or activator protein 1 (AP-1)] are also required for the transcriptional activation of IL-2, IFN-γ, IL-4, IL-6, and TNF-α in T cells. “Others” denotes other critical transcription factors (table S1). NF-κB is required for TCR-induced production of multiple cytokines; however, the GLK–IKKβ–NF-κB cascade alone is not sufficient for the induction of multiple cytokines. Collectively, GLK overexpression or TCR signaling induces IL-17A transcription through AhR and RORγt in T cells.
    Anti Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8036 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp gapdh hs02758991 g1
    TCR signaling induces RORγt phosphorylation and subsequent <t>AhR-RORγt</t> interaction. ( A ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, p-IKKβ (Ser 180/181 ), and IKKβ in primary splenic T cells. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( B ) Coimmunoprecipitation of endogenous AhR with RORγt from lysates of murine primary splenic T cells stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( C ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, and IKKβ in primary splenic T cells of IKKβ f/f or CD4-Cre;IKKβ f/f mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( D ) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt (left) or between AhR and Ser 489 -phosphorylated RORγt (right) in primary T cells of IKKβ f/f or IKKβ f/f ;CD4-Cre mice. T cells were stimulated as in (C). Each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Original magnification, ×630; scale bars, 10 μm. ( E and F ) ELISA of various cytokines in supernatants of primary splenic T cells from IKKβ f/f or IKKβ f/f ;CD4-Cre mice (E), as well as RORγt f/f or RORγt f/f ;CD4-Cre mice (F). T cells were stimulated with plate-bound anti-CD3 antibodies (2 μg each per milliliter) for 3 days. Means ± SD are shown. n = 3 per group. ( G ) Immunoblotting of RORγt and <t>GAPDH</t> proteins from primary splenic T cells of RORγt f/f or RORγt f/f ;CD4-Cre mice. Data shown (A to G) are representative of three independent experiments. ( H ) Schematic model of IL-17A transcription induced by the AhR-RORγt complex in GLK-overexpressing or TCR-stimulated T cells. GLK overexpression in T cells of T cell–specific GLK Tg (Lck-GLK Tg) mice induces AhR Ser 36 phosphorylation through PKCθ and also induces RORγt Ser 489 phosphorylation through IKKβ. Once RORγt is phosphorylated, RORγt interacts directly with AhR. Phosphorylated AhR is responsible for transporting RORγt into cell nucleus. The AhR-RORγt complex binds to both the RORγt-binding element (−877 to −872) and the AhR-binding element (−254 to −249) of the IL-17A promoter, leading to induction of IL-17A transcription. In normal T cells, TCR stimulation also induces GLK kinase activity and downstream signaling, including IKKβ activation, RORγt Ser 489 phosphorylation, and the AhR-RORγt interaction. Besides NF-κB, other critical transcription factors [such as nuclear factor of activated T cell 1 (NFAT1) or activator protein 1 (AP-1)] are also required for the transcriptional activation of IL-2, IFN-γ, IL-4, IL-6, and TNF-α in T cells. “Others” denotes other critical transcription factors (table S1). NF-κB is required for TCR-induced production of multiple cytokines; however, the GLK–IKKβ–NF-κB cascade alone is not sufficient for the induction of multiple cytokines. Collectively, GLK overexpression or TCR signaling induces IL-17A transcription through AhR and RORγt in T cells.
    Gene Exp Gapdh Hs02758991 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4697 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti gapdh
    TCR signaling induces RORγt phosphorylation and subsequent <t>AhR-RORγt</t> interaction. ( A ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, p-IKKβ (Ser 180/181 ), and IKKβ in primary splenic T cells. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( B ) Coimmunoprecipitation of endogenous AhR with RORγt from lysates of murine primary splenic T cells stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( C ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, and IKKβ in primary splenic T cells of IKKβ f/f or CD4-Cre;IKKβ f/f mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( D ) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt (left) or between AhR and Ser 489 -phosphorylated RORγt (right) in primary T cells of IKKβ f/f or IKKβ f/f ;CD4-Cre mice. T cells were stimulated as in (C). Each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Original magnification, ×630; scale bars, 10 μm. ( E and F ) ELISA of various cytokines in supernatants of primary splenic T cells from IKKβ f/f or IKKβ f/f ;CD4-Cre mice (E), as well as RORγt f/f or RORγt f/f ;CD4-Cre mice (F). T cells were stimulated with plate-bound anti-CD3 antibodies (2 μg each per milliliter) for 3 days. Means ± SD are shown. n = 3 per group. ( G ) Immunoblotting of RORγt and <t>GAPDH</t> proteins from primary splenic T cells of RORγt f/f or RORγt f/f ;CD4-Cre mice. Data shown (A to G) are representative of three independent experiments. ( H ) Schematic model of IL-17A transcription induced by the AhR-RORγt complex in GLK-overexpressing or TCR-stimulated T cells. GLK overexpression in T cells of T cell–specific GLK Tg (Lck-GLK Tg) mice induces AhR Ser 36 phosphorylation through PKCθ and also induces RORγt Ser 489 phosphorylation through IKKβ. Once RORγt is phosphorylated, RORγt interacts directly with AhR. Phosphorylated AhR is responsible for transporting RORγt into cell nucleus. The AhR-RORγt complex binds to both the RORγt-binding element (−877 to −872) and the AhR-binding element (−254 to −249) of the IL-17A promoter, leading to induction of IL-17A transcription. In normal T cells, TCR stimulation also induces GLK kinase activity and downstream signaling, including IKKβ activation, RORγt Ser 489 phosphorylation, and the AhR-RORγt interaction. Besides NF-κB, other critical transcription factors [such as nuclear factor of activated T cell 1 (NFAT1) or activator protein 1 (AP-1)] are also required for the transcriptional activation of IL-2, IFN-γ, IL-4, IL-6, and TNF-α in T cells. “Others” denotes other critical transcription factors (table S1). NF-κB is required for TCR-induced production of multiple cytokines; however, the GLK–IKKβ–NF-κB cascade alone is not sufficient for the induction of multiple cytokines. Collectively, GLK overexpression or TCR signaling induces IL-17A transcription through AhR and RORγt in T cells.
    Mouse Anti Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti gapdh antibody
    TCR signaling induces RORγt phosphorylation and subsequent <t>AhR-RORγt</t> interaction. ( A ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, p-IKKβ (Ser 180/181 ), and IKKβ in primary splenic T cells. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( B ) Coimmunoprecipitation of endogenous AhR with RORγt from lysates of murine primary splenic T cells stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( C ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, and IKKβ in primary splenic T cells of IKKβ f/f or CD4-Cre;IKKβ f/f mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( D ) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt (left) or between AhR and Ser 489 -phosphorylated RORγt (right) in primary T cells of IKKβ f/f or IKKβ f/f ;CD4-Cre mice. T cells were stimulated as in (C). Each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Original magnification, ×630; scale bars, 10 μm. ( E and F ) ELISA of various cytokines in supernatants of primary splenic T cells from IKKβ f/f or IKKβ f/f ;CD4-Cre mice (E), as well as RORγt f/f or RORγt f/f ;CD4-Cre mice (F). T cells were stimulated with plate-bound anti-CD3 antibodies (2 μg each per milliliter) for 3 days. Means ± SD are shown. n = 3 per group. ( G ) Immunoblotting of RORγt and <t>GAPDH</t> proteins from primary splenic T cells of RORγt f/f or RORγt f/f ;CD4-Cre mice. Data shown (A to G) are representative of three independent experiments. ( H ) Schematic model of IL-17A transcription induced by the AhR-RORγt complex in GLK-overexpressing or TCR-stimulated T cells. GLK overexpression in T cells of T cell–specific GLK Tg (Lck-GLK Tg) mice induces AhR Ser 36 phosphorylation through PKCθ and also induces RORγt Ser 489 phosphorylation through IKKβ. Once RORγt is phosphorylated, RORγt interacts directly with AhR. Phosphorylated AhR is responsible for transporting RORγt into cell nucleus. The AhR-RORγt complex binds to both the RORγt-binding element (−877 to −872) and the AhR-binding element (−254 to −249) of the IL-17A promoter, leading to induction of IL-17A transcription. In normal T cells, TCR stimulation also induces GLK kinase activity and downstream signaling, including IKKβ activation, RORγt Ser 489 phosphorylation, and the AhR-RORγt interaction. Besides NF-κB, other critical transcription factors [such as nuclear factor of activated T cell 1 (NFAT1) or activator protein 1 (AP-1)] are also required for the transcriptional activation of IL-2, IFN-γ, IL-4, IL-6, and TNF-α in T cells. “Others” denotes other critical transcription factors (table S1). NF-κB is required for TCR-induced production of multiple cytokines; however, the GLK–IKKβ–NF-κB cascade alone is not sufficient for the induction of multiple cytokines. Collectively, GLK overexpression or TCR signaling induces IL-17A transcription through AhR and RORγt in T cells.
    Anti Gapdh Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti gapdh antibody mouse monoclonal
    TCR signaling induces RORγt phosphorylation and subsequent <t>AhR-RORγt</t> interaction. ( A ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, p-IKKβ (Ser 180/181 ), and IKKβ in primary splenic T cells. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( B ) Coimmunoprecipitation of endogenous AhR with RORγt from lysates of murine primary splenic T cells stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( C ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, and IKKβ in primary splenic T cells of IKKβ f/f or CD4-Cre;IKKβ f/f mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( D ) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt (left) or between AhR and Ser 489 -phosphorylated RORγt (right) in primary T cells of IKKβ f/f or IKKβ f/f ;CD4-Cre mice. T cells were stimulated as in (C). Each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Original magnification, ×630; scale bars, 10 μm. ( E and F ) ELISA of various cytokines in supernatants of primary splenic T cells from IKKβ f/f or IKKβ f/f ;CD4-Cre mice (E), as well as RORγt f/f or RORγt f/f ;CD4-Cre mice (F). T cells were stimulated with plate-bound anti-CD3 antibodies (2 μg each per milliliter) for 3 days. Means ± SD are shown. n = 3 per group. ( G ) Immunoblotting of RORγt and <t>GAPDH</t> proteins from primary splenic T cells of RORγt f/f or RORγt f/f ;CD4-Cre mice. Data shown (A to G) are representative of three independent experiments. ( H ) Schematic model of IL-17A transcription induced by the AhR-RORγt complex in GLK-overexpressing or TCR-stimulated T cells. GLK overexpression in T cells of T cell–specific GLK Tg (Lck-GLK Tg) mice induces AhR Ser 36 phosphorylation through PKCθ and also induces RORγt Ser 489 phosphorylation through IKKβ. Once RORγt is phosphorylated, RORγt interacts directly with AhR. Phosphorylated AhR is responsible for transporting RORγt into cell nucleus. The AhR-RORγt complex binds to both the RORγt-binding element (−877 to −872) and the AhR-binding element (−254 to −249) of the IL-17A promoter, leading to induction of IL-17A transcription. In normal T cells, TCR stimulation also induces GLK kinase activity and downstream signaling, including IKKβ activation, RORγt Ser 489 phosphorylation, and the AhR-RORγt interaction. Besides NF-κB, other critical transcription factors [such as nuclear factor of activated T cell 1 (NFAT1) or activator protein 1 (AP-1)] are also required for the transcriptional activation of IL-2, IFN-γ, IL-4, IL-6, and TNF-α in T cells. “Others” denotes other critical transcription factors (table S1). NF-κB is required for TCR-induced production of multiple cytokines; however, the GLK–IKKβ–NF-κB cascade alone is not sufficient for the induction of multiple cytokines. Collectively, GLK overexpression or TCR signaling induces IL-17A transcription through AhR and RORγt in T cells.
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    Santa Cruz Biotechnology glyceraldehyde 3 phosphate dehydrogenase gapdh
    Wnt5a expressing tumors have less Wnt/β-catenin signaling than MMTV-Wnt1 tumors. (A) Quantitative RT-PCR of Wnt/ β -catenin target genes . Expression of Axin2 mRNA in MMTV-Wnt1 versus MMTV-Wnt1;MMTV-Wnt5a tumors as determined by quantitative RT-PCR (n = 5 MMTV-Wnt1, n = 5 MMTV-Wnt1;MMTV-Wnt5a). Data are shown as tables obtained using REST software. Axin2 mRNA was significantly down-regulated in MMTV-Wnt1;MMTV-Wnt5a tumors. (B) Western blot for β-catenin protein . Protein lysates were prepared from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a tumors. β-catenin and glyceraldehyde 3-phosphate dehydrogenase <t>(GAPDH)</t> were used as loading controls. The ratio of active β-catenin to β-catenin as determined by densitometic analysis is shown. MMTV-Wnt1;MMTV-Wnt5a tumors displayed decreased levels of active β-catenin compared to controls.
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    Wnt5a expressing tumors have less Wnt/β-catenin signaling than MMTV-Wnt1 tumors. (A) Quantitative RT-PCR of Wnt/ β -catenin target genes . Expression of Axin2 mRNA in MMTV-Wnt1 versus MMTV-Wnt1;MMTV-Wnt5a tumors as determined by quantitative RT-PCR (n = 5 MMTV-Wnt1, n = 5 MMTV-Wnt1;MMTV-Wnt5a). Data are shown as tables obtained using REST software. Axin2 mRNA was significantly down-regulated in MMTV-Wnt1;MMTV-Wnt5a tumors. (B) Western blot for β-catenin protein . Protein lysates were prepared from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a tumors. β-catenin and glyceraldehyde 3-phosphate dehydrogenase <t>(GAPDH)</t> were used as loading controls. The ratio of active β-catenin to β-catenin as determined by densitometic analysis is shown. MMTV-Wnt1;MMTV-Wnt5a tumors displayed decreased levels of active β-catenin compared to controls.
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    Proteintech mouse gapdh monoclonal
    Wnt5a expressing tumors have less Wnt/β-catenin signaling than MMTV-Wnt1 tumors. (A) Quantitative RT-PCR of Wnt/ β -catenin target genes . Expression of Axin2 mRNA in MMTV-Wnt1 versus MMTV-Wnt1;MMTV-Wnt5a tumors as determined by quantitative RT-PCR (n = 5 MMTV-Wnt1, n = 5 MMTV-Wnt1;MMTV-Wnt5a). Data are shown as tables obtained using REST software. Axin2 mRNA was significantly down-regulated in MMTV-Wnt1;MMTV-Wnt5a tumors. (B) Western blot for β-catenin protein . Protein lysates were prepared from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a tumors. β-catenin and glyceraldehyde 3-phosphate dehydrogenase <t>(GAPDH)</t> were used as loading controls. The ratio of active β-catenin to β-catenin as determined by densitometic analysis is shown. MMTV-Wnt1;MMTV-Wnt5a tumors displayed decreased levels of active β-catenin compared to controls.
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    Image Search Results


    TRBP Phosphorylation Reduces Merlin Binding and TRBP Polyubiquitination (A) Representative immunoblot (left) and HMW TRBP protein levels, quantified from immunoblot of lysates from hippocampal neurons treated with vehicle (DMSO) or MG132 (60 min), normalized to GAPDH and plotted relative to vehicle alone (set as 1.0). (B) Representative immunoblot (left) and quantification of TRBP protein bound to GST-S5a (ubiquitin binding motif) (right) using lysates from hippocampal neurons pre-treated with either U0126 or vehicle control (30 min), followed by BDNF (60 min). Ubiquitinated TRBP protein was normalized to total ubiquitin pull-down and plotted relative to vehicle (set as 1.0). *non-TRBP specific band reactive with secondary antibody. (C) Lysates from HEK293T cells co-expressing FL-TRBPWT, SΔA, SΔD, or PCDNA3.1 alone (control) with HA-K48 ubiquitin underwent stringent IP with anti-FLAG antibody, followed by immunoblot with anti-FLAG and anti-HA antibodies. Shown are representative immunoblots (left) and quantification (right) of co-associated HA-K48 ubiquitin normalized to the amount of immunoprecipitated FL-TRBP for each construct, plotted relative to the FL-TRBPWT condition (set as 1.0). (D) Lysates from HEK293T cells co-expressing FL-TRBPWT, SΔA, SΔD, or PCDNA3.1 alone (control) with HA-Merlin were immunoprecipitated with anti-FLAG antibody, followed by immunoblot with anti-FLAG and anti-HA antibodies. Shown are representative immunoblots (left) and quantification (right) of co-associated HA-Merlin protein normalized first to the amount of input HA-Merlin protein and next to the immunoprecipitated FL-TRBP for each construct, plotted relative to the FL-TRBPWT condition (set as 1.0). .

    Journal: Molecular cell

    Article Title: A Rapid Induction Mechanism for Lin28a in Trophic Responses

    doi: 10.1016/j.molcel.2016.12.025

    Figure Lengend Snippet: TRBP Phosphorylation Reduces Merlin Binding and TRBP Polyubiquitination (A) Representative immunoblot (left) and HMW TRBP protein levels, quantified from immunoblot of lysates from hippocampal neurons treated with vehicle (DMSO) or MG132 (60 min), normalized to GAPDH and plotted relative to vehicle alone (set as 1.0). (B) Representative immunoblot (left) and quantification of TRBP protein bound to GST-S5a (ubiquitin binding motif) (right) using lysates from hippocampal neurons pre-treated with either U0126 or vehicle control (30 min), followed by BDNF (60 min). Ubiquitinated TRBP protein was normalized to total ubiquitin pull-down and plotted relative to vehicle (set as 1.0). *non-TRBP specific band reactive with secondary antibody. (C) Lysates from HEK293T cells co-expressing FL-TRBPWT, SΔA, SΔD, or PCDNA3.1 alone (control) with HA-K48 ubiquitin underwent stringent IP with anti-FLAG antibody, followed by immunoblot with anti-FLAG and anti-HA antibodies. Shown are representative immunoblots (left) and quantification (right) of co-associated HA-K48 ubiquitin normalized to the amount of immunoprecipitated FL-TRBP for each construct, plotted relative to the FL-TRBPWT condition (set as 1.0). (D) Lysates from HEK293T cells co-expressing FL-TRBPWT, SΔA, SΔD, or PCDNA3.1 alone (control) with HA-Merlin were immunoprecipitated with anti-FLAG antibody, followed by immunoblot with anti-FLAG and anti-HA antibodies. Shown are representative immunoblots (left) and quantification (right) of co-associated HA-Merlin protein normalized first to the amount of input HA-Merlin protein and next to the immunoprecipitated FL-TRBP for each construct, plotted relative to the FL-TRBPWT condition (set as 1.0). .

    Article Snippet: The following reverse transcription program was used: 1) 37°C incubation for 60 min, 2) 95°C inactivation for 5 min. Products of RT reaction were then assayed via qPCR using TaqMan mRNA qPCR probes: Lin28a (Applied Biosystems Mm00524077_m1), β-tubulin 3 (Applied Biosystems Mm00727586_s1), and GAPDH (Applied Bio-systems Mm99999915_g1).

    Techniques: Binding Assay, Expressing, Western Blot, Immunoprecipitation, Construct

    BDNF Post-translationally Induces Lin28a, but Not Paralog Lin28b, through Protein Stabilization (A) Quantification (top) and representative immunoblot (bottom) of Lin28a protein levels in lysates from hippocampal neurons exposed to BDNF with or without transcription blockade (Actinomycin-D) for the indicated times, normalized to GAPDH and plotted relative to 0-min BDNF without Actino-mycin-D (set as 1.0). #p

    Journal: Molecular cell

    Article Title: A Rapid Induction Mechanism for Lin28a in Trophic Responses

    doi: 10.1016/j.molcel.2016.12.025

    Figure Lengend Snippet: BDNF Post-translationally Induces Lin28a, but Not Paralog Lin28b, through Protein Stabilization (A) Quantification (top) and representative immunoblot (bottom) of Lin28a protein levels in lysates from hippocampal neurons exposed to BDNF with or without transcription blockade (Actinomycin-D) for the indicated times, normalized to GAPDH and plotted relative to 0-min BDNF without Actino-mycin-D (set as 1.0). #p

    Article Snippet: The following reverse transcription program was used: 1) 37°C incubation for 60 min, 2) 95°C inactivation for 5 min. Products of RT reaction were then assayed via qPCR using TaqMan mRNA qPCR probes: Lin28a (Applied Biosystems Mm00524077_m1), β-tubulin 3 (Applied Biosystems Mm00727586_s1), and GAPDH (Applied Bio-systems Mm99999915_g1).

    Techniques:

    Western blot analysis of transforming growth factor beta (TGF‐β) and alpha‐smooth muscle actin (α‐SMA). (a) Cytoplasmic fractions were analyzed by WB with TGF‐β and α‐SMA and glyceraldehyde phosphate dehydrogenase (GAPDH) antibodies. (b) Arbitrary values expressed as mean and SD. * P

    Journal: JGH Open: An Open Access Journal of Gastroenterology and Hepatology

    Article Title: Antifibrogenic effect of melatonin in rats with experimental liver cirrhosis induced by carbon tetrachloride

    doi: 10.1002/jgh3.12055

    Figure Lengend Snippet: Western blot analysis of transforming growth factor beta (TGF‐β) and alpha‐smooth muscle actin (α‐SMA). (a) Cytoplasmic fractions were analyzed by WB with TGF‐β and α‐SMA and glyceraldehyde phosphate dehydrogenase (GAPDH) antibodies. (b) Arbitrary values expressed as mean and SD. * P

    Article Snippet: The anti‐β‐actin (A5060/42 kDa) and GAPDH (G9545/37 kDa) antibody (Sigma Aldrich) was at 1:2000 dilution with TTBS in 5% nonfat dry milk.

    Techniques: Western Blot

    Inhibition of clathrin-mediated endocytosis does not affect mitochondrial morphology a , Representative images of TOM20 immunofluorescence in n = 50, 50, 52 COS-7 cells transfected with scrambled control, AP-2, or Dyn2 siRNA. Scale bars = 10 μm. b , Immuno-blots with antibodies against AP-2, Dyn-2, and GAPDH in siRNA-treated cells. c , The effect on mitochondrial morphology was quantitated within a 230 μm 2 region of interest (ROI) for mean area per mitochondria (left graph), and mean mitochondria per ROI (right graph). As in , Dyn2–depleted cells had larger mitochondria and less mitochondrion per ROI compared to control cells; however, AP-2 depleted cells displayed mitochondrial morphology that was qualitatively and quantitatively similar to control cells. These data were obtained from three biological replicate experiments. Error bars represent the s.e.m. *p

    Journal: Nature

    Article Title: Multiple Dynamin family members collaborate to drive mitochondrial division

    doi: 10.1038/nature20555

    Figure Lengend Snippet: Inhibition of clathrin-mediated endocytosis does not affect mitochondrial morphology a , Representative images of TOM20 immunofluorescence in n = 50, 50, 52 COS-7 cells transfected with scrambled control, AP-2, or Dyn2 siRNA. Scale bars = 10 μm. b , Immuno-blots with antibodies against AP-2, Dyn-2, and GAPDH in siRNA-treated cells. c , The effect on mitochondrial morphology was quantitated within a 230 μm 2 region of interest (ROI) for mean area per mitochondria (left graph), and mean mitochondria per ROI (right graph). As in , Dyn2–depleted cells had larger mitochondria and less mitochondrion per ROI compared to control cells; however, AP-2 depleted cells displayed mitochondrial morphology that was qualitatively and quantitatively similar to control cells. These data were obtained from three biological replicate experiments. Error bars represent the s.e.m. *p

    Article Snippet: Protein levels in whole-cell lysates and cell fractions were assayed by Western blot with polyclonal rabbit antibodies against Dyn2 (ab3457, Abcam), phosphoSerine637-Drp1 (4867S, Cell Signaling Tech), phosphoSerine616-Drp1 (3455S, Cell Signaling Tech), Mff (17090-1-AP, Protein Tech), Mfn2 clone 4H8 (WH0009927M3, Sigma-Aldrich), α-Tubulin (ab18251, Abcam), TOM20 (sc-11415, Santa Cruz Biotech), Bax (sc-493, Santa Cruz Biotech), GAPDH (G9545, Sigma-Aldrich), and the monoclonal mouse antibody against AP-1/2 (sc-17771, Santa Cruz Biotech.), Drp1 (ab56788, Abcam), Dynamin (610245, BD Biosciences), cytochrome C (sc-13560, Santa Cruz Biotech), Opa1 (612606, BD Biosciences).

    Techniques: Inhibition, Immunofluorescence, Transfection, Western Blot

    Mapping ELK1 polypeptide segments required for co-activation by AR(A/B) by internal deletion analysis. A shows data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream Gal4 elements ( GAL4-TATA-LUC ) and also with a vector expressing the AR A/B domain fused to the VP16 transactivation domain. Cells were transfected with plasmids expressing Gal4 fusion proteins of ELK1. The fusion constructs substituted the Gal4 DNA binding domain ( Gal4-DBD ) for the ETS DNA binding domain of ELK1. Within this fusion construct, a series of internal deletions were made, as indicated in the schematic in A . Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The promoter activity shown on the y axis required the presence of the AR A/B domain because knocking down AR(A/B) expression in the same cells transfected with full-length Gal4-ELK1 decreased the promoter activity to the basal value shown in the figure for Gal4-DBD alone. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). B shows data obtained using recombinant HeLa cells generated by stably transducing only GAL4-TATA-LUC . The cells were transfected with each of the Gal4-ELK1 fusion constructs used in A and co-transfected with an expression plasmid for a constitutively active mutant of MEK1 or with the vector control. Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). C shows a schematic of the domain organization of ELK1; here, the deletion mapping of two ELK1 polypeptide segments encompassing residues required for association with AR(A/B) (data from Figs. 3 A , 4 A, and 5 A ) is represented by gray shading of the two segments. For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. In all panels, the error bars represent standard deviation of experimental triplicates. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth *

    doi: 10.1074/jbc.M116.745596

    Figure Lengend Snippet: Mapping ELK1 polypeptide segments required for co-activation by AR(A/B) by internal deletion analysis. A shows data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream Gal4 elements ( GAL4-TATA-LUC ) and also with a vector expressing the AR A/B domain fused to the VP16 transactivation domain. Cells were transfected with plasmids expressing Gal4 fusion proteins of ELK1. The fusion constructs substituted the Gal4 DNA binding domain ( Gal4-DBD ) for the ETS DNA binding domain of ELK1. Within this fusion construct, a series of internal deletions were made, as indicated in the schematic in A . Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The promoter activity shown on the y axis required the presence of the AR A/B domain because knocking down AR(A/B) expression in the same cells transfected with full-length Gal4-ELK1 decreased the promoter activity to the basal value shown in the figure for Gal4-DBD alone. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). B shows data obtained using recombinant HeLa cells generated by stably transducing only GAL4-TATA-LUC . The cells were transfected with each of the Gal4-ELK1 fusion constructs used in A and co-transfected with an expression plasmid for a constitutively active mutant of MEK1 or with the vector control. Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). C shows a schematic of the domain organization of ELK1; here, the deletion mapping of two ELK1 polypeptide segments encompassing residues required for association with AR(A/B) (data from Figs. 3 A , 4 A, and 5 A ) is represented by gray shading of the two segments. For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. In all panels, the error bars represent standard deviation of experimental triplicates. *, p

    Article Snippet: Affinity-purified rabbit anti-human antibody to AR (sc-7305) and mouse antibodies to Gal4 (sc-510) and GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Activation Assay, Recombinant, Generated, Stable Transfection, Luciferase, Plasmid Preparation, Expressing, Transfection, Construct, Binding Assay, Activity Assay, Western Blot, Mutagenesis, Standard Deviation

    Functional association of ELK1 and AR-V7 and effect on cell growth. A, HeLa cells were co-transfected with an ELK1-driven minimal promoter-luciferase reporter (( ELK1 ) 2 - TATA-LUC ) and expression plasmid for either AR-V7 or WTELK1 or control vector plasmid for 48 h. Luciferase activity was measured in the cell lysates. For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. B, HeLa cells were co-transfected with a Gal4-driven minimal promoter-luciferase reporter (Gal4-TATA-Luc) and expression plasmid for either AR-V7 or Gal4-ELK1 or control vector plasmid for 48 h. Luciferase activity was measured in the cell lysates. For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. C shows a Western blot of HeLa cell lysates corresponding to all of the transfections in A and B, which was probed using an antibody to the amino-terminal domain of AR or with antibody to GAPDH (loading control). D, top panel shows the effect of depleting ELK1 by lentiviral shRNA transduction on the growth of CWR22Rv1 cells monitored by the MTT assay compared with control shRNA. The Western blot in the bottom panel shows ELK1 shRNA-induced depletion of ELK1 compared with control shRNA; GAPDH was probed as the loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth *

    doi: 10.1074/jbc.M116.745596

    Figure Lengend Snippet: Functional association of ELK1 and AR-V7 and effect on cell growth. A, HeLa cells were co-transfected with an ELK1-driven minimal promoter-luciferase reporter (( ELK1 ) 2 - TATA-LUC ) and expression plasmid for either AR-V7 or WTELK1 or control vector plasmid for 48 h. Luciferase activity was measured in the cell lysates. For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. B, HeLa cells were co-transfected with a Gal4-driven minimal promoter-luciferase reporter (Gal4-TATA-Luc) and expression plasmid for either AR-V7 or Gal4-ELK1 or control vector plasmid for 48 h. Luciferase activity was measured in the cell lysates. For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. C shows a Western blot of HeLa cell lysates corresponding to all of the transfections in A and B, which was probed using an antibody to the amino-terminal domain of AR or with antibody to GAPDH (loading control). D, top panel shows the effect of depleting ELK1 by lentiviral shRNA transduction on the growth of CWR22Rv1 cells monitored by the MTT assay compared with control shRNA. The Western blot in the bottom panel shows ELK1 shRNA-induced depletion of ELK1 compared with control shRNA; GAPDH was probed as the loading control.

    Article Snippet: Affinity-purified rabbit anti-human antibody to AR (sc-7305) and mouse antibodies to Gal4 (sc-510) and GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Functional Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Western Blot, shRNA, Transduction, MTT Assay

    Further refinement of the mapping of ELK1 motifs required for co-activation by AR(A/B). A shows the ELK1 polypeptide sequence. The bracketed segments indicate the two segments that were mapped from the deletion analyses in Figs. 3 – 5 as regions containing residues essential for the association of ELK1 with AR(A/B). The boxed segments denote the D-domain of ELK1 and the F X FP motif of ELK1. The segments in bold font represent peptides that were deleted for further mapping in B and C. B and D show data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream Gal4 elements ( GAL4-TATA-LUC ) and also with a vector expressing the AR A/B domain fused to the VP16 transactivation domain. Cells were transfected with plasmids expressing Gal4 fusion proteins of ELK1. The fusion constructs substituted the Gal4 DNA binding domain ( Gal4-DBD ) for the ETS DNA binding domain of ELK1. Within this fusion construct, the indicated internal deletions or mutations were made, as indicated in the schematics in B and D . Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The promoter activity shown on the y axis required the presence of the AR A/B domain as knocking down AR(A/B) expression in the same cells transfected with full-length Gal4-ELK1 decreased the promoter activity to the basal value shown in the figure for Gal4-DBD alone. The insets in B and D show cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). C and E show data obtained using recombinant HeLa cells generated by stably transducing only GAL4-TATA-LUC . The cells were transfected with each of the Gal4-ELK1 fusion constructs used in B and D , respectively, and co-transfected with an expression plasmid for a constitutively active mutant of MEK1 or with the vector control. Forty eight hours after transfection with the various Gal4-ELK1fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The insets in C and E show cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. In all panels, the error bars represent standard deviation of experimental triplicates. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth *

    doi: 10.1074/jbc.M116.745596

    Figure Lengend Snippet: Further refinement of the mapping of ELK1 motifs required for co-activation by AR(A/B). A shows the ELK1 polypeptide sequence. The bracketed segments indicate the two segments that were mapped from the deletion analyses in Figs. 3 – 5 as regions containing residues essential for the association of ELK1 with AR(A/B). The boxed segments denote the D-domain of ELK1 and the F X FP motif of ELK1. The segments in bold font represent peptides that were deleted for further mapping in B and C. B and D show data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream Gal4 elements ( GAL4-TATA-LUC ) and also with a vector expressing the AR A/B domain fused to the VP16 transactivation domain. Cells were transfected with plasmids expressing Gal4 fusion proteins of ELK1. The fusion constructs substituted the Gal4 DNA binding domain ( Gal4-DBD ) for the ETS DNA binding domain of ELK1. Within this fusion construct, the indicated internal deletions or mutations were made, as indicated in the schematics in B and D . Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The promoter activity shown on the y axis required the presence of the AR A/B domain as knocking down AR(A/B) expression in the same cells transfected with full-length Gal4-ELK1 decreased the promoter activity to the basal value shown in the figure for Gal4-DBD alone. The insets in B and D show cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). C and E show data obtained using recombinant HeLa cells generated by stably transducing only GAL4-TATA-LUC . The cells were transfected with each of the Gal4-ELK1 fusion constructs used in B and D , respectively, and co-transfected with an expression plasmid for a constitutively active mutant of MEK1 or with the vector control. Forty eight hours after transfection with the various Gal4-ELK1fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The insets in C and E show cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. In all panels, the error bars represent standard deviation of experimental triplicates. *, p

    Article Snippet: Affinity-purified rabbit anti-human antibody to AR (sc-7305) and mouse antibodies to Gal4 (sc-510) and GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Activation Assay, Sequencing, Recombinant, Generated, Stable Transfection, Luciferase, Plasmid Preparation, Expressing, Transfection, Construct, Binding Assay, Activity Assay, Western Blot, Mutagenesis, Standard Deviation

    Mapping ELK1 polypeptide segments required for co-activation by AR(A/B) by amino-terminal deletion analysis. A shows data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream Gal4 elements ( GAL4-TATA-LUC ) and also with a vector expressing the AR A/B domain fused to the VP16 transactivation domain. Cells were transfected with plasmids expressing Gal4 fusion proteins of ELK1. The fusion constructs substituted the Gal4 DNA binding domain (Gal4-DBD) for the ETS DNA binding domain of ELK1. Within this fusion construct, a series of amino-terminal deletions were made, as indicated in the schematic in A . Forty eight hours after transfection with the various Gal4-ELK1fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The promoter activity shown on the y axis required the presence of the AR A/B domain as knocking down AR(A/B) expression in the same cells transfected with full-length Gal4-ELK1 decreased the promoter activity to the basal value shown in the figure for Gal4-DBD alone. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). B shows data obtained using recombinant HeLa cells generated by stably transducing only GAL4-TATA-LUC . The cells were transfected with each of the Gal4-ELK1 fusion constructs used in A and co-transfected with an expression plasmid for a constitutively active mutant of MEK1 or with the vector control. Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). C shows a schematic of the domain organization of ELK1; here, the amino-terminal deletion mapping of an ELK1 polypeptide segment encompassing residues required for association with AR(A/B) (data from A ) is represented by gray shading . For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. In all panels, the error bars represent standard deviation of experimental triplicates. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth *

    doi: 10.1074/jbc.M116.745596

    Figure Lengend Snippet: Mapping ELK1 polypeptide segments required for co-activation by AR(A/B) by amino-terminal deletion analysis. A shows data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream Gal4 elements ( GAL4-TATA-LUC ) and also with a vector expressing the AR A/B domain fused to the VP16 transactivation domain. Cells were transfected with plasmids expressing Gal4 fusion proteins of ELK1. The fusion constructs substituted the Gal4 DNA binding domain (Gal4-DBD) for the ETS DNA binding domain of ELK1. Within this fusion construct, a series of amino-terminal deletions were made, as indicated in the schematic in A . Forty eight hours after transfection with the various Gal4-ELK1fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The promoter activity shown on the y axis required the presence of the AR A/B domain as knocking down AR(A/B) expression in the same cells transfected with full-length Gal4-ELK1 decreased the promoter activity to the basal value shown in the figure for Gal4-DBD alone. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). B shows data obtained using recombinant HeLa cells generated by stably transducing only GAL4-TATA-LUC . The cells were transfected with each of the Gal4-ELK1 fusion constructs used in A and co-transfected with an expression plasmid for a constitutively active mutant of MEK1 or with the vector control. Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). C shows a schematic of the domain organization of ELK1; here, the amino-terminal deletion mapping of an ELK1 polypeptide segment encompassing residues required for association with AR(A/B) (data from A ) is represented by gray shading . For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. In all panels, the error bars represent standard deviation of experimental triplicates. *, p

    Article Snippet: Affinity-purified rabbit anti-human antibody to AR (sc-7305) and mouse antibodies to Gal4 (sc-510) and GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Activation Assay, Recombinant, Generated, Stable Transfection, Luciferase, Plasmid Preparation, Expressing, Transfection, Construct, Binding Assay, Activity Assay, Western Blot, Mutagenesis, Standard Deviation

    Effect of depleting SRF or ERK1/2 on the interactions of AR with ELK1. A and B show data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream Gal4 elements ( GAL4-TATA-LUC ) and also with vectors expressing AR and a Gal4-ELK1 fusion construct in which the Gal4 DNA binding domain was substituted for the ETS DNA binding domain of ELK1. The cells were depleted of hormone and then transduced with shRNA against SRF or a non-targeted control shRNA using lentivirus. Seventy two hours after infection, cells were treated with testosterone (10 n m ) or vehicle for a further 48 h. The cells were then harvested to quantify SRF mRNA ( A ) or for Western blotting analysis using antibody to SRF or to GAPDH (loading control) ( A, inset ) or for luciferase activity ( B ). C and D show data obtained using recombinant HeLa cells generated by stably transducing GAL4-TATA-LUC and also a vector expressing the AR A/B domain fused to the VP16 transactivation domain (AR(A/B)-VP16). The cells were transduced with shRNA against SRF or a non-targeted control shRNA using lentivirus. Seventy two hours after infection, the cells were transfected with expression plasmid for the Gal4-ELK1 fusion protein. Forty eight hours later, the cells were harvested to quantify SRF mRNA ( C ) or for Western blotting analysis using antibody to SRF or to GAPDH (loading control) ( C, inset ) or to measure luciferase activity ( D ). E–G show data obtained using the recombinant HeLa cells with stably incorporated GAL4-TATA-LUC and stably expressing the AR(A/B)-VP16 fusion protein. Cells were transfected with a mixture of siRNA against ERK1 and ERK2 or with control non-targeted siRNA. After 48 h of transfection, the cells were harvested to quantify mRNAs for ERK1 and ERK2 ( E ) or for Western blotting analysis using antibody to ERK1/2 or to GAPDH (loading control) ( E, below ); the remaining cells were transfected for a second time with the Gal-4ELK1 expression plasmid or the control vector plasmid or the plasmid for constitutively active mutant of MEK1 ( F ). After a further 24 h, the cells were harvested to quantify mRNAs for ERK1 and ERK2 ( E ) or for Western blotting analysis using antibody to ERK1/2 or to GAPDH (loading control) ( E, below ) or to measure luciferase activity ( F ). H, HeLa cells were co-transfected with an ELK1-driven minimal promoter-luciferase reporter (( ELK1 ) 2 - TATA-LUC ) and expression plasmid for either AR(A/B) or constitutively active MEK1 or control vector plasmid. The cells were treated with trametinib (1 μ m ) or vehicle for 48 h beginning with the time of transfection. Luciferase activity was measured in the cell lysates. For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. In all panels, the error bars represent standard deviation of experimental triplicates. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth *

    doi: 10.1074/jbc.M116.745596

    Figure Lengend Snippet: Effect of depleting SRF or ERK1/2 on the interactions of AR with ELK1. A and B show data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream Gal4 elements ( GAL4-TATA-LUC ) and also with vectors expressing AR and a Gal4-ELK1 fusion construct in which the Gal4 DNA binding domain was substituted for the ETS DNA binding domain of ELK1. The cells were depleted of hormone and then transduced with shRNA against SRF or a non-targeted control shRNA using lentivirus. Seventy two hours after infection, cells were treated with testosterone (10 n m ) or vehicle for a further 48 h. The cells were then harvested to quantify SRF mRNA ( A ) or for Western blotting analysis using antibody to SRF or to GAPDH (loading control) ( A, inset ) or for luciferase activity ( B ). C and D show data obtained using recombinant HeLa cells generated by stably transducing GAL4-TATA-LUC and also a vector expressing the AR A/B domain fused to the VP16 transactivation domain (AR(A/B)-VP16). The cells were transduced with shRNA against SRF or a non-targeted control shRNA using lentivirus. Seventy two hours after infection, the cells were transfected with expression plasmid for the Gal4-ELK1 fusion protein. Forty eight hours later, the cells were harvested to quantify SRF mRNA ( C ) or for Western blotting analysis using antibody to SRF or to GAPDH (loading control) ( C, inset ) or to measure luciferase activity ( D ). E–G show data obtained using the recombinant HeLa cells with stably incorporated GAL4-TATA-LUC and stably expressing the AR(A/B)-VP16 fusion protein. Cells were transfected with a mixture of siRNA against ERK1 and ERK2 or with control non-targeted siRNA. After 48 h of transfection, the cells were harvested to quantify mRNAs for ERK1 and ERK2 ( E ) or for Western blotting analysis using antibody to ERK1/2 or to GAPDH (loading control) ( E, below ); the remaining cells were transfected for a second time with the Gal-4ELK1 expression plasmid or the control vector plasmid or the plasmid for constitutively active mutant of MEK1 ( F ). After a further 24 h, the cells were harvested to quantify mRNAs for ERK1 and ERK2 ( E ) or for Western blotting analysis using antibody to ERK1/2 or to GAPDH (loading control) ( E, below ) or to measure luciferase activity ( F ). H, HeLa cells were co-transfected with an ELK1-driven minimal promoter-luciferase reporter (( ELK1 ) 2 - TATA-LUC ) and expression plasmid for either AR(A/B) or constitutively active MEK1 or control vector plasmid. The cells were treated with trametinib (1 μ m ) or vehicle for 48 h beginning with the time of transfection. Luciferase activity was measured in the cell lysates. For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. In all panels, the error bars represent standard deviation of experimental triplicates. *, p

    Article Snippet: Affinity-purified rabbit anti-human antibody to AR (sc-7305) and mouse antibodies to Gal4 (sc-510) and GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Recombinant, Generated, Stable Transfection, Luciferase, Expressing, Construct, Binding Assay, Transduction, shRNA, Infection, Western Blot, Activity Assay, Plasmid Preparation, Transfection, Mutagenesis, Standard Deviation

    ELK1 motifs required for co-activation by full-length AR. A shows data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream Gal4 elements ( GAL4-TATA-LUC ). Cells were plated in hormone-depleted media and co-transfected with expression plasmids for the indicated Gal4-ELK1 fusion proteins and an expression plasmid for full-length AR. The fusion constructs substituted the Gal4 DNA binding domain (Gal4-DBD) for the ETS DNA binding domain of ELK1. At the time of transfection, the cells were treated with testosterone (10 n m ) or vehicle control. Forty eight hours after transfection, cells were harvested by preparing lysates for measurement of luciferase activity. B shows cell lysates probed by Western blotting with antibodies against Gal4, AR, or GAPDH (loading control). C shows data on co-immunoprecipitation of ectopic AR and ectopic ELK1 or ELK1 mutants from HeLa cell lysates. HeLa cells were transfected with the expression plasmid for AR and co-transfected with an expression plasmid for WTELK1 or one of two ELK1 mutants, ELK1Δ308–321 and ELK1 FxLa. The lysates were immunoprecipitated ( IP ) using either antibody to AR or a negative control. The immunoprecipitates were probed by Western blotting using antibody to either AR or ELK1 as indicated. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth *

    doi: 10.1074/jbc.M116.745596

    Figure Lengend Snippet: ELK1 motifs required for co-activation by full-length AR. A shows data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream Gal4 elements ( GAL4-TATA-LUC ). Cells were plated in hormone-depleted media and co-transfected with expression plasmids for the indicated Gal4-ELK1 fusion proteins and an expression plasmid for full-length AR. The fusion constructs substituted the Gal4 DNA binding domain (Gal4-DBD) for the ETS DNA binding domain of ELK1. At the time of transfection, the cells were treated with testosterone (10 n m ) or vehicle control. Forty eight hours after transfection, cells were harvested by preparing lysates for measurement of luciferase activity. B shows cell lysates probed by Western blotting with antibodies against Gal4, AR, or GAPDH (loading control). C shows data on co-immunoprecipitation of ectopic AR and ectopic ELK1 or ELK1 mutants from HeLa cell lysates. HeLa cells were transfected with the expression plasmid for AR and co-transfected with an expression plasmid for WTELK1 or one of two ELK1 mutants, ELK1Δ308–321 and ELK1 FxLa. The lysates were immunoprecipitated ( IP ) using either antibody to AR or a negative control. The immunoprecipitates were probed by Western blotting using antibody to either AR or ELK1 as indicated. *, p

    Article Snippet: Affinity-purified rabbit anti-human antibody to AR (sc-7305) and mouse antibodies to Gal4 (sc-510) and GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Activation Assay, Recombinant, Generated, Stable Transfection, Luciferase, Transfection, Expressing, Plasmid Preparation, Construct, Binding Assay, Activity Assay, Western Blot, Immunoprecipitation, Negative Control

    Mapping ELK1 polypeptide segments required for co-activation by AR(A/B) by carboxyl-terminal deletion analysis. A shows data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream Gal4 elements ( GAL4-TATA-LUC ) and also with a vector expressing the AR A/B domain fused to the VP16 transactivation domain. Cells were transfected with plasmids expressing Gal4 fusion proteins of ELK1. The fusion constructs substituted the Gal4 DNA binding domain ( Gal4-DBD ) for the ETS DNA binding domain of ELK1. Within this fusion construct, a series of carboxyl-terminal deletions were made, as indicated in the schematic in A . Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The promoter activity shown on the y axis required the presence of the AR A/B domain because knocking down AR(A/B) expression in the same cells transfected with full-length Gal4-ELK1 decreased the promoter activity to the basal value shown in the figure for Gal4-DBD alone. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). B shows data obtained using recombinant HeLa cells generated by stably transducing only GAL4-TATA-LUC . The cells were transfected with each of the Gal4-ELK1 fusion constructs used in A and co-transfected with an expression plasmid for a constitutively active mutant of MEK1 or with the vector control. Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). C shows a schematic of the domain organization of ELK1; here, the carboxyl-terminal deletion mapping of an ELK1 polypeptide segment encompassing residues required for association with AR(A/B) (data from A ) is represented by gray shading . For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. In all panels, the error bars represent standard deviation of experimental triplicates. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth *

    doi: 10.1074/jbc.M116.745596

    Figure Lengend Snippet: Mapping ELK1 polypeptide segments required for co-activation by AR(A/B) by carboxyl-terminal deletion analysis. A shows data obtained using recombinant HeLa cells generated by stably transducing a minimal promoter-luciferase reporter containing upstream Gal4 elements ( GAL4-TATA-LUC ) and also with a vector expressing the AR A/B domain fused to the VP16 transactivation domain. Cells were transfected with plasmids expressing Gal4 fusion proteins of ELK1. The fusion constructs substituted the Gal4 DNA binding domain ( Gal4-DBD ) for the ETS DNA binding domain of ELK1. Within this fusion construct, a series of carboxyl-terminal deletions were made, as indicated in the schematic in A . Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The promoter activity shown on the y axis required the presence of the AR A/B domain because knocking down AR(A/B) expression in the same cells transfected with full-length Gal4-ELK1 decreased the promoter activity to the basal value shown in the figure for Gal4-DBD alone. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). B shows data obtained using recombinant HeLa cells generated by stably transducing only GAL4-TATA-LUC . The cells were transfected with each of the Gal4-ELK1 fusion constructs used in A and co-transfected with an expression plasmid for a constitutively active mutant of MEK1 or with the vector control. Forty eight hours after transfection with the various Gal4-ELK1 fusion constructs, cells were harvested by preparing lysates for measurement of luciferase activity. The inset shows cell lysates probed by Western blotting with antibodies against Gal4 or GAPDH (loading control). C shows a schematic of the domain organization of ELK1; here, the carboxyl-terminal deletion mapping of an ELK1 polypeptide segment encompassing residues required for association with AR(A/B) (data from A ) is represented by gray shading . For all transfections, a Renilla luciferase reporter was used as the control for transfection efficiency. In all panels, the error bars represent standard deviation of experimental triplicates. *, p

    Article Snippet: Affinity-purified rabbit anti-human antibody to AR (sc-7305) and mouse antibodies to Gal4 (sc-510) and GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Activation Assay, Recombinant, Generated, Stable Transfection, Luciferase, Plasmid Preparation, Expressing, Transfection, Construct, Binding Assay, Activity Assay, Western Blot, Mutagenesis, Standard Deviation

    Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of IκBα and p-IκBα protein; (D-F) Expression of nuclear and cytosolic p65 protein. Histone H3 served as the nuclear loading control, GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P

    Journal: Oncology Reports

    Article Title: Xanthohumol, a prenylated flavonoid from Hops, exerts anticancer effects against gastric cancer in vitro

    doi: 10.3892/or.2018.6723

    Figure Lengend Snippet: Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of IκBα and p-IκBα protein; (D-F) Expression of nuclear and cytosolic p65 protein. Histone H3 served as the nuclear loading control, GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P

    Article Snippet: Antibodies against Bcl-2 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab194583), Bax (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32503), p-IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab133462), IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32518), p65 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab16502), histone H3 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab1791) and GAPDH (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab9485) were obtained from Abcam (Cambridge, UK).

    Techniques: Western Blot, Expressing

    Effect of xanthohumol (Xn) on the nuclear factor (NF)-κB signaling pathway in AGS cells. Cells were treated with different concentrations of Xn (0–20 µ M) for 24 h, then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Effects of Xn on IκBα and p-IκBα expression. (D-F) Effect of Xn on nuclear and cytosolic p65 expression. Histone H3 served as the nuclear loading control and GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. *P

    Journal: Oncology Reports

    Article Title: Xanthohumol, a prenylated flavonoid from Hops, exerts anticancer effects against gastric cancer in vitro

    doi: 10.3892/or.2018.6723

    Figure Lengend Snippet: Effect of xanthohumol (Xn) on the nuclear factor (NF)-κB signaling pathway in AGS cells. Cells were treated with different concentrations of Xn (0–20 µ M) for 24 h, then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Effects of Xn on IκBα and p-IκBα expression. (D-F) Effect of Xn on nuclear and cytosolic p65 expression. Histone H3 served as the nuclear loading control and GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. *P

    Article Snippet: Antibodies against Bcl-2 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab194583), Bax (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32503), p-IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab133462), IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32518), p65 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab16502), histone H3 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab1791) and GAPDH (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab9485) were obtained from Abcam (Cambridge, UK).

    Techniques: Western Blot, Expressing

    PKCε protein levels in HeLaPKCεA/E and glioblastoma cell lines. Protein extracts from HeLaPKCεA/E (−Dox), HeLaPKCεA/E (+Dox), U-118 MG, U-138 MG, T98G, LN18 cell lines were subjected to Western blot analysis using antibodies against PKCε and GAPDH. HeLaPKCεA/E cells with doxycycline-induced expression of PKCε (+Dox) and without doxycycline treatment (−Dox) served as a reference cell lines with low and high expression of PKCε, respectively. The average relative absorbance of three independent experiments is presented in a bar graph

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: PKCε protein levels in HeLaPKCεA/E and glioblastoma cell lines. Protein extracts from HeLaPKCεA/E (−Dox), HeLaPKCεA/E (+Dox), U-118 MG, U-138 MG, T98G, LN18 cell lines were subjected to Western blot analysis using antibodies against PKCε and GAPDH. HeLaPKCεA/E cells with doxycycline-induced expression of PKCε (+Dox) and without doxycycline treatment (−Dox) served as a reference cell lines with low and high expression of PKCε, respectively. The average relative absorbance of three independent experiments is presented in a bar graph

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Western Blot, Expressing

    Knockdown of PKCɛ diminishes the level and phosphorylation of Akt in glioma cells. a U-138 MG and b U-118 MG were transfected for 72 h with PKCε siRNA and Control siRNA and then were treated for 24 h with rapamycin (300 nM) or 3-MA (5 mM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Knockdown of PKCɛ diminishes the level and phosphorylation of Akt in glioma cells. a U-138 MG and b U-118 MG were transfected for 72 h with PKCε siRNA and Control siRNA and then were treated for 24 h with rapamycin (300 nM) or 3-MA (5 mM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection

    Influence of PKCε downregulation and rapamycin or 3-MA treatment on protein level directly involved in autophagy activation (mTOR, PI3K, Beclin1). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. In case of mTOR the order of bands has been changing, due to a different final construct of probe layout. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Influence of PKCε downregulation and rapamycin or 3-MA treatment on protein level directly involved in autophagy activation (mTOR, PI3K, Beclin1). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. In case of mTOR the order of bands has been changing, due to a different final construct of probe layout. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Activation Assay, Transfection, Construct

    Effect of PKCε downregulation and rapamycin or 3-MA treatment on proteins engaged in the formation of a double-membrane structure known as autophagosome (Atg5, LC3-II). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation and rapamycin or 3-MA treatment on proteins engaged in the formation of a double-membrane structure known as autophagosome (Atg5, LC3-II). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection

    Effect of PKCε downregulation and rapamycin or 3-MA treatment on Bcl-2, a protein that regulates autophagy process. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation and rapamycin or 3-MA treatment on Bcl-2, a protein that regulates autophagy process. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection

    Effect of PKCε downregulation on the adhesion of glioblastoma cells. a . U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and Control siRNA and then were seeded in culture plates coated with Matrigel (Corning Life Sciences, NY, USA). The photos represent cell adhesion under the microscope at 100 x magnification field. c Evaluation of FAK expression in U-138 MG and U-118 MG cells with knockdown PKCε. Total protein expression and FAK phosphorylation at Tyr-397/Tyr-576/577 were assessed. GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation on the adhesion of glioblastoma cells. a . U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and Control siRNA and then were seeded in culture plates coated with Matrigel (Corning Life Sciences, NY, USA). The photos represent cell adhesion under the microscope at 100 x magnification field. c Evaluation of FAK expression in U-138 MG and U-118 MG cells with knockdown PKCε. Total protein expression and FAK phosphorylation at Tyr-397/Tyr-576/577 were assessed. GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection, Microscopy, Expressing

    Modulation SQSTM1/p62 after PRKCE silencing and rapamycin treatment. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Modulation SQSTM1/p62 after PRKCE silencing and rapamycin treatment. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection

    CERS6 binds to intracellular domains of CD95/Fas. a Constructs (pCMV6-mycDDK or pCMV6-AC-HA) encoding a human wild-type CERS6 tagged with mycDDK at COOH terminus, FAS full-length and FAS mutants tagged with HA epitopes. HD homeobox (DNA binding) domain, TLC TRAM/LAG/CLN8 homology domain, CRD cysteine-rich domain, TD transmembrane domain, DD death domain. b Co-immunoprecipitation of full-length CERS6 and Fas and Fas mutants to detect direct binding. c Co-immunoprecipitation of full-length CERS6 and Fas/Fas mutants in cellular fractions (Cyt cytosolic fraction that includes ER and other organelles, PM plasma membrane). GAPDH (right) is used as a marker for cytosolic fraction while E-cadherin is the marker for plasma membrane

    Journal: Cell Death & Disease

    Article Title: Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia

    doi: 10.1038/s41419-018-0964-4

    Figure Lengend Snippet: CERS6 binds to intracellular domains of CD95/Fas. a Constructs (pCMV6-mycDDK or pCMV6-AC-HA) encoding a human wild-type CERS6 tagged with mycDDK at COOH terminus, FAS full-length and FAS mutants tagged with HA epitopes. HD homeobox (DNA binding) domain, TLC TRAM/LAG/CLN8 homology domain, CRD cysteine-rich domain, TD transmembrane domain, DD death domain. b Co-immunoprecipitation of full-length CERS6 and Fas and Fas mutants to detect direct binding. c Co-immunoprecipitation of full-length CERS6 and Fas/Fas mutants in cellular fractions (Cyt cytosolic fraction that includes ER and other organelles, PM plasma membrane). GAPDH (right) is used as a marker for cytosolic fraction while E-cadherin is the marker for plasma membrane

    Article Snippet: Chemicals and reagents Sphingolipid standards includingC14:0-, C16:0-, C17:0-, C18:0-, C18:1-, C20:0-, C24:0-, C24:1-ceramide and C18:0-, C18:1-, C24:0-, C24:1-dihydroceramide were purchased from Avanti Polar Lipids (Alabaster, AL); ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburg, PA); chloroform, ethyl acetate, methanol, 2-propanol, NaF, NaHCO3 , Na3 VO4 , Tris-HCl, Triton X-100, pepstatin A, aprotinin, leupeptin, 200 proof ethanol, isopropanol, puromycin, dexamethasone, and anti-FLAG-M2 (1 μg/ml) antibody from Sigma-Aldrich (St. Louis, MO); ABT-737 from Cayman Chemical (Ann Arbor, MI); DTT, EDTA, NaCl, PMSF, SDS, TBE, trypsin/EDTA, Lipofectamine®, PLUSTM reagent, Superscript® III first-strand synthesis system for RT-PCR from Thermo Fisher Scientific (Waltham, MA); Triton X-114 from Acros Organics (Morris, NJ); Z-IETD from R & D Systems (Minneapolis, MN); Fas ligand from GeneTex (Irvine, CA); anti-CERS6, anti-FLIP and anti-GAPDH antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fas and anti-FADD from BD Transduction Laboratories (San Jose, CA); anti-HA antibody from Roche (Indianapolis, IN); anti-caspase-3, anti-cleaved-caspase-3, anti-caspase-8 and anti-PARP from Cell Signaling Technology (Danvers, MA); Age 1-HF, Bam H1-HF, Eco R1-HF, Mlu 1-HF, Pme 1 and Sgf 1 restriction enzymes from New England Biolabs (Ipswich, MA); bovine serum albumin from Jackson ImmunoResearch Laboratories (West Grove, PA); all oligos were synthesized from Integrated DNA Technologies (Coralville, IA).

    Techniques: Construct, Binding Assay, Thin Layer Chromatography, Immunoprecipitation, Marker

    CERS6 is overexpressed in ALL. a Biosynthesis and metabolism of ceramides. Ceramides are generated either de novo or via the salvage pathway. They are metabolized to sphingosine or may serve as substrates for the synthesis of glucosyl ceramides, lactosyl ceramides or sphingomyelins. Sphingomyelins degrade to ceramides via the sphingomyelinase pathway and are synthesized from ceramides via sphingomyelin synthase. b mRNA expression of the six ceramide synthase isoforms in the NCI PPTP panel of 23 cell lines comprising six different pediatric cancers. RBD rhabdomyosarcoma, BT brain tumor, EFT Ewing’s family of tumors, NB neuroblastoma, ALL acute lymphoblastic leukemia, LYM lymphoma. The mRNA expression was determined within the NCI Pediatric Preclinical Testing Program. c CERS6 protein expression in acute lymphoblastic leukemia (ALL) cell lines in comparison to peripheral blood mononuclear cells (PBMCs) and T lymphocytes obtained from blood of healthy human volunteers. GAPDH was used as a loading control. d C 16 -Ceramides in T-cell ALL cells in comparison to PBMCs and T lymphocytes. Ceramide levels were quantitated by HPLC/MS/MS (PBMC: 0.28 ± 0.04, n = 18; T lymphocytes: 0.36 ± 0.01, n = 3; T-ALL: 0.54 ± 0.09, n = 9). e CERS6 protein expression in T lymphocytes isolated from primary lymphoid malignancy samples in comparison to normal T lymphocytes *** p

    Journal: Cell Death & Disease

    Article Title: Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia

    doi: 10.1038/s41419-018-0964-4

    Figure Lengend Snippet: CERS6 is overexpressed in ALL. a Biosynthesis and metabolism of ceramides. Ceramides are generated either de novo or via the salvage pathway. They are metabolized to sphingosine or may serve as substrates for the synthesis of glucosyl ceramides, lactosyl ceramides or sphingomyelins. Sphingomyelins degrade to ceramides via the sphingomyelinase pathway and are synthesized from ceramides via sphingomyelin synthase. b mRNA expression of the six ceramide synthase isoforms in the NCI PPTP panel of 23 cell lines comprising six different pediatric cancers. RBD rhabdomyosarcoma, BT brain tumor, EFT Ewing’s family of tumors, NB neuroblastoma, ALL acute lymphoblastic leukemia, LYM lymphoma. The mRNA expression was determined within the NCI Pediatric Preclinical Testing Program. c CERS6 protein expression in acute lymphoblastic leukemia (ALL) cell lines in comparison to peripheral blood mononuclear cells (PBMCs) and T lymphocytes obtained from blood of healthy human volunteers. GAPDH was used as a loading control. d C 16 -Ceramides in T-cell ALL cells in comparison to PBMCs and T lymphocytes. Ceramide levels were quantitated by HPLC/MS/MS (PBMC: 0.28 ± 0.04, n = 18; T lymphocytes: 0.36 ± 0.01, n = 3; T-ALL: 0.54 ± 0.09, n = 9). e CERS6 protein expression in T lymphocytes isolated from primary lymphoid malignancy samples in comparison to normal T lymphocytes *** p

    Article Snippet: Chemicals and reagents Sphingolipid standards includingC14:0-, C16:0-, C17:0-, C18:0-, C18:1-, C20:0-, C24:0-, C24:1-ceramide and C18:0-, C18:1-, C24:0-, C24:1-dihydroceramide were purchased from Avanti Polar Lipids (Alabaster, AL); ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburg, PA); chloroform, ethyl acetate, methanol, 2-propanol, NaF, NaHCO3 , Na3 VO4 , Tris-HCl, Triton X-100, pepstatin A, aprotinin, leupeptin, 200 proof ethanol, isopropanol, puromycin, dexamethasone, and anti-FLAG-M2 (1 μg/ml) antibody from Sigma-Aldrich (St. Louis, MO); ABT-737 from Cayman Chemical (Ann Arbor, MI); DTT, EDTA, NaCl, PMSF, SDS, TBE, trypsin/EDTA, Lipofectamine®, PLUSTM reagent, Superscript® III first-strand synthesis system for RT-PCR from Thermo Fisher Scientific (Waltham, MA); Triton X-114 from Acros Organics (Morris, NJ); Z-IETD from R & D Systems (Minneapolis, MN); Fas ligand from GeneTex (Irvine, CA); anti-CERS6, anti-FLIP and anti-GAPDH antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fas and anti-FADD from BD Transduction Laboratories (San Jose, CA); anti-HA antibody from Roche (Indianapolis, IN); anti-caspase-3, anti-cleaved-caspase-3, anti-caspase-8 and anti-PARP from Cell Signaling Technology (Danvers, MA); Age 1-HF, Bam H1-HF, Eco R1-HF, Mlu 1-HF, Pme 1 and Sgf 1 restriction enzymes from New England Biolabs (Ipswich, MA); bovine serum albumin from Jackson ImmunoResearch Laboratories (West Grove, PA); all oligos were synthesized from Integrated DNA Technologies (Coralville, IA).

    Techniques: Generated, Synthesized, Expressing, High Performance Liquid Chromatography, Mass Spectrometry, Isolation

    ALL cells are sensitized to ABT-737 upon CERS6 knockdown. a CERS6 protein levels in CCRF-CEM and MOLT-4 cells after stably knocking down CERS6 using shRNA (NT-shRNA non-targeted shRNA, sh CERS6 -Mixed shRNA against CERS6 and selected with puromycin, sh CERS6 -Single shRNA against CERS6 and selected with puromycin followed by repopulation from a single cell). GAPDH was used as a loading control. b C 16 -Ceramide levels (pmole/nmole of inorganic phosphate) in ALL cells after CERS6 knockdown. Ceramide levels were quantitated by HPLC/MS/MS (1.5 ± 0.1 vs 7.3 ± 0.9 pmole/nmole Pi for CCRF-CEM and 7.4 ± 0.4 vs 10.9 ± 0.3 pmole/nmole Pi for MOLT-4; n = 3). c Knockdown of CERS6 in CCRF-CEM and MOLT-4 sensitized the cells to ABT-737. Dose response curves showing concentration of ABT-737 on x -axis and survival fraction on y -axis on a log 10 scale. Bar graphs depict survival fractions at 100 nM of ABT-737 in both cell lines (5.2 ± 2.7% vs 86.6 ± 4.6% survival at 100 nM ABT-737 for CCRF-CEM and 26.1 ± 3.8% vs 73.6 ± 4.6% survival at 100 nM ABT-737 for MOLT-4; n = 6). d Annexin-V apoptosis assay by flow cytometry showing the percentage of live and apoptotic cells upon ABT-737 treatment (1 µM for 16 h) in CCRF-CEM or MOLT-4 cells with CERS6 knockdown in comparison to cells transduced with NT-shRNA (98.0 ± 0.4% vs 38.0 ± 0.4% for CCRF-CEM and 90.8 ± 0.3% vs 72.1 ± 0.3% for MOLT-4; n = 3). e CERS6 knockdown in CCRF-CEM or MOLT-4 cells show higher levels of cleaved PARP and cleaved caspase-3 on ABT-737 treatment (1 µM for 16 h). GAPDH was used as a loading control * p

    Journal: Cell Death & Disease

    Article Title: Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia

    doi: 10.1038/s41419-018-0964-4

    Figure Lengend Snippet: ALL cells are sensitized to ABT-737 upon CERS6 knockdown. a CERS6 protein levels in CCRF-CEM and MOLT-4 cells after stably knocking down CERS6 using shRNA (NT-shRNA non-targeted shRNA, sh CERS6 -Mixed shRNA against CERS6 and selected with puromycin, sh CERS6 -Single shRNA against CERS6 and selected with puromycin followed by repopulation from a single cell). GAPDH was used as a loading control. b C 16 -Ceramide levels (pmole/nmole of inorganic phosphate) in ALL cells after CERS6 knockdown. Ceramide levels were quantitated by HPLC/MS/MS (1.5 ± 0.1 vs 7.3 ± 0.9 pmole/nmole Pi for CCRF-CEM and 7.4 ± 0.4 vs 10.9 ± 0.3 pmole/nmole Pi for MOLT-4; n = 3). c Knockdown of CERS6 in CCRF-CEM and MOLT-4 sensitized the cells to ABT-737. Dose response curves showing concentration of ABT-737 on x -axis and survival fraction on y -axis on a log 10 scale. Bar graphs depict survival fractions at 100 nM of ABT-737 in both cell lines (5.2 ± 2.7% vs 86.6 ± 4.6% survival at 100 nM ABT-737 for CCRF-CEM and 26.1 ± 3.8% vs 73.6 ± 4.6% survival at 100 nM ABT-737 for MOLT-4; n = 6). d Annexin-V apoptosis assay by flow cytometry showing the percentage of live and apoptotic cells upon ABT-737 treatment (1 µM for 16 h) in CCRF-CEM or MOLT-4 cells with CERS6 knockdown in comparison to cells transduced with NT-shRNA (98.0 ± 0.4% vs 38.0 ± 0.4% for CCRF-CEM and 90.8 ± 0.3% vs 72.1 ± 0.3% for MOLT-4; n = 3). e CERS6 knockdown in CCRF-CEM or MOLT-4 cells show higher levels of cleaved PARP and cleaved caspase-3 on ABT-737 treatment (1 µM for 16 h). GAPDH was used as a loading control * p

    Article Snippet: Chemicals and reagents Sphingolipid standards includingC14:0-, C16:0-, C17:0-, C18:0-, C18:1-, C20:0-, C24:0-, C24:1-ceramide and C18:0-, C18:1-, C24:0-, C24:1-dihydroceramide were purchased from Avanti Polar Lipids (Alabaster, AL); ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburg, PA); chloroform, ethyl acetate, methanol, 2-propanol, NaF, NaHCO3 , Na3 VO4 , Tris-HCl, Triton X-100, pepstatin A, aprotinin, leupeptin, 200 proof ethanol, isopropanol, puromycin, dexamethasone, and anti-FLAG-M2 (1 μg/ml) antibody from Sigma-Aldrich (St. Louis, MO); ABT-737 from Cayman Chemical (Ann Arbor, MI); DTT, EDTA, NaCl, PMSF, SDS, TBE, trypsin/EDTA, Lipofectamine®, PLUSTM reagent, Superscript® III first-strand synthesis system for RT-PCR from Thermo Fisher Scientific (Waltham, MA); Triton X-114 from Acros Organics (Morris, NJ); Z-IETD from R & D Systems (Minneapolis, MN); Fas ligand from GeneTex (Irvine, CA); anti-CERS6, anti-FLIP and anti-GAPDH antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fas and anti-FADD from BD Transduction Laboratories (San Jose, CA); anti-HA antibody from Roche (Indianapolis, IN); anti-caspase-3, anti-cleaved-caspase-3, anti-caspase-8 and anti-PARP from Cell Signaling Technology (Danvers, MA); Age 1-HF, Bam H1-HF, Eco R1-HF, Mlu 1-HF, Pme 1 and Sgf 1 restriction enzymes from New England Biolabs (Ipswich, MA); bovine serum albumin from Jackson ImmunoResearch Laboratories (West Grove, PA); all oligos were synthesized from Integrated DNA Technologies (Coralville, IA).

    Techniques: Stable Transfection, shRNA, High Performance Liquid Chromatography, Mass Spectrometry, Concentration Assay, Apoptosis Assay, Flow Cytometry, Cytometry, Transduction

    CERS6 alters ALL cells sensitivity to ABT-737 via the extrinsic pathway of apoptosis which can be overcome by a caspase-8 inhibitor. a Higher caspase-8 activity seen with CCRF-CEM and MOLT-4 CERS6 knockdown cells in comparison to cells transduced with non-targeted shRNA upon ABT-737 treatment. GAPDH was used as a loading control. b Annexin-V apoptosis assay by flow cytometry showing the percentage of apoptotic cells in ABT-737-treated CERS6 knockdown cells (CCRF-CEM and MOLT-4), with or without caspase-8 inhibitor, Z-IETD (23.4 ± 1.7% vs 37.5 ± 1.5% for CCRF-CEM and 8.2 ± 0.1% vs 20.8 ± 0.3% for MOLT-4; n = 3). c ABT-737-treated CERS6 knockdown cells show decreased levels of cleaved Caspase-8, cleaved PARP and cleaved caspase-3 on addition of Z-IETD in comparison to ABT-737-treated CERS6 knockdown cells without Z-IETD. d Levels of soluble FasL released in cultured medium upon treatment of ABT-737 in CERS6 knockdown cells compared to cells transduced with non-targeted shRNA. e Left: Caspase-8 activity was increased in a dose-dependent manner upon treatment of varying concentrations of Fas ligand (FasL) in CCRF-CEM cells with CERS6 knockdown. GAPDH was used as a loading control. e Right: Annexin-V apoptosis assay by flow cytometry showing increase in apoptotic cells in CCRF-CEM cells with CERS6 knockdown in comparison to cells transduced with NT-shRNA upon treatment of 1000 ng/ml of FasL (70.9 ± 0.6% vs 14.1 ± 0.1%; n = 3) ** p

    Journal: Cell Death & Disease

    Article Title: Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia

    doi: 10.1038/s41419-018-0964-4

    Figure Lengend Snippet: CERS6 alters ALL cells sensitivity to ABT-737 via the extrinsic pathway of apoptosis which can be overcome by a caspase-8 inhibitor. a Higher caspase-8 activity seen with CCRF-CEM and MOLT-4 CERS6 knockdown cells in comparison to cells transduced with non-targeted shRNA upon ABT-737 treatment. GAPDH was used as a loading control. b Annexin-V apoptosis assay by flow cytometry showing the percentage of apoptotic cells in ABT-737-treated CERS6 knockdown cells (CCRF-CEM and MOLT-4), with or without caspase-8 inhibitor, Z-IETD (23.4 ± 1.7% vs 37.5 ± 1.5% for CCRF-CEM and 8.2 ± 0.1% vs 20.8 ± 0.3% for MOLT-4; n = 3). c ABT-737-treated CERS6 knockdown cells show decreased levels of cleaved Caspase-8, cleaved PARP and cleaved caspase-3 on addition of Z-IETD in comparison to ABT-737-treated CERS6 knockdown cells without Z-IETD. d Levels of soluble FasL released in cultured medium upon treatment of ABT-737 in CERS6 knockdown cells compared to cells transduced with non-targeted shRNA. e Left: Caspase-8 activity was increased in a dose-dependent manner upon treatment of varying concentrations of Fas ligand (FasL) in CCRF-CEM cells with CERS6 knockdown. GAPDH was used as a loading control. e Right: Annexin-V apoptosis assay by flow cytometry showing increase in apoptotic cells in CCRF-CEM cells with CERS6 knockdown in comparison to cells transduced with NT-shRNA upon treatment of 1000 ng/ml of FasL (70.9 ± 0.6% vs 14.1 ± 0.1%; n = 3) ** p

    Article Snippet: Chemicals and reagents Sphingolipid standards includingC14:0-, C16:0-, C17:0-, C18:0-, C18:1-, C20:0-, C24:0-, C24:1-ceramide and C18:0-, C18:1-, C24:0-, C24:1-dihydroceramide were purchased from Avanti Polar Lipids (Alabaster, AL); ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburg, PA); chloroform, ethyl acetate, methanol, 2-propanol, NaF, NaHCO3 , Na3 VO4 , Tris-HCl, Triton X-100, pepstatin A, aprotinin, leupeptin, 200 proof ethanol, isopropanol, puromycin, dexamethasone, and anti-FLAG-M2 (1 μg/ml) antibody from Sigma-Aldrich (St. Louis, MO); ABT-737 from Cayman Chemical (Ann Arbor, MI); DTT, EDTA, NaCl, PMSF, SDS, TBE, trypsin/EDTA, Lipofectamine®, PLUSTM reagent, Superscript® III first-strand synthesis system for RT-PCR from Thermo Fisher Scientific (Waltham, MA); Triton X-114 from Acros Organics (Morris, NJ); Z-IETD from R & D Systems (Minneapolis, MN); Fas ligand from GeneTex (Irvine, CA); anti-CERS6, anti-FLIP and anti-GAPDH antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fas and anti-FADD from BD Transduction Laboratories (San Jose, CA); anti-HA antibody from Roche (Indianapolis, IN); anti-caspase-3, anti-cleaved-caspase-3, anti-caspase-8 and anti-PARP from Cell Signaling Technology (Danvers, MA); Age 1-HF, Bam H1-HF, Eco R1-HF, Mlu 1-HF, Pme 1 and Sgf 1 restriction enzymes from New England Biolabs (Ipswich, MA); bovine serum albumin from Jackson ImmunoResearch Laboratories (West Grove, PA); all oligos were synthesized from Integrated DNA Technologies (Coralville, IA).

    Techniques: Activity Assay, Transduction, shRNA, Apoptosis Assay, Flow Cytometry, Cytometry, Cell Culture

    ALL cells overexpressing CERS6 show resistance to ABT-737. a CERS6 levels in CCRF-CEM cells upon CERS6 overexpression (Control cells transduced with empty vector, CERS6 -Mixed CERS6 overexpressing cells and selected with puromycin, CERS6 -Single CERS6 overexpressing cells and selected with puromycin followed by repopulation from a single cell; 10.1 ± 0.5 vs 5.7 ± 0.1 pmole/nmole Pi; n = 3). b C 16 -Cer levels (pmole/nmole of inorganic phosphate) in CCRF-CEM cells after CERS6 overexpression. Ceramide levels were quantitated by HPLC/MS/MS. c CERS6 overexpression in CCRF-CEM cells rendered the cells resistant to ABT-737. Dose response curves showing concentration of ABT-737 on x -axis and survival fraction on y -axis on a log 10 scale. Bar graphs depict survival fractions at 300 nM of ABT-737.(39.5 ± 2.2% vs 2.3 ± 1.8% survival at 300 nM ABT-737; n = 6) ( d ) Annexin-V apoptosis assay by flow cytometry showing the percentage of live and apoptotic cells upon ABT-737 treatment (2 µM for 16 h) in CERS6 overexpressing CCRF-CEM cells in comparison to cells transduced with empty vector (36.8 ± 3.8% vs 85.2 ± 0.6%; n = 3). e CERS6 overexpressing CCRF-CEM cells show lower levels of cleaved PARP and cleaved Caspase-3 on ABT-737 treatment (2 µM for 16 h) in comparison to control cells. The far right lane (sh CERS6 -Single) is a sample from ABT-737-treated CERS6 knockdown CCRF-CEM cells (from Fig. 2e ) and represents positive control for cleaved PARP and cleaved caspase-3. GAPDH was used as a loading control * p

    Journal: Cell Death & Disease

    Article Title: Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia

    doi: 10.1038/s41419-018-0964-4

    Figure Lengend Snippet: ALL cells overexpressing CERS6 show resistance to ABT-737. a CERS6 levels in CCRF-CEM cells upon CERS6 overexpression (Control cells transduced with empty vector, CERS6 -Mixed CERS6 overexpressing cells and selected with puromycin, CERS6 -Single CERS6 overexpressing cells and selected with puromycin followed by repopulation from a single cell; 10.1 ± 0.5 vs 5.7 ± 0.1 pmole/nmole Pi; n = 3). b C 16 -Cer levels (pmole/nmole of inorganic phosphate) in CCRF-CEM cells after CERS6 overexpression. Ceramide levels were quantitated by HPLC/MS/MS. c CERS6 overexpression in CCRF-CEM cells rendered the cells resistant to ABT-737. Dose response curves showing concentration of ABT-737 on x -axis and survival fraction on y -axis on a log 10 scale. Bar graphs depict survival fractions at 300 nM of ABT-737.(39.5 ± 2.2% vs 2.3 ± 1.8% survival at 300 nM ABT-737; n = 6) ( d ) Annexin-V apoptosis assay by flow cytometry showing the percentage of live and apoptotic cells upon ABT-737 treatment (2 µM for 16 h) in CERS6 overexpressing CCRF-CEM cells in comparison to cells transduced with empty vector (36.8 ± 3.8% vs 85.2 ± 0.6%; n = 3). e CERS6 overexpressing CCRF-CEM cells show lower levels of cleaved PARP and cleaved Caspase-3 on ABT-737 treatment (2 µM for 16 h) in comparison to control cells. The far right lane (sh CERS6 -Single) is a sample from ABT-737-treated CERS6 knockdown CCRF-CEM cells (from Fig. 2e ) and represents positive control for cleaved PARP and cleaved caspase-3. GAPDH was used as a loading control * p

    Article Snippet: Chemicals and reagents Sphingolipid standards includingC14:0-, C16:0-, C17:0-, C18:0-, C18:1-, C20:0-, C24:0-, C24:1-ceramide and C18:0-, C18:1-, C24:0-, C24:1-dihydroceramide were purchased from Avanti Polar Lipids (Alabaster, AL); ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburg, PA); chloroform, ethyl acetate, methanol, 2-propanol, NaF, NaHCO3 , Na3 VO4 , Tris-HCl, Triton X-100, pepstatin A, aprotinin, leupeptin, 200 proof ethanol, isopropanol, puromycin, dexamethasone, and anti-FLAG-M2 (1 μg/ml) antibody from Sigma-Aldrich (St. Louis, MO); ABT-737 from Cayman Chemical (Ann Arbor, MI); DTT, EDTA, NaCl, PMSF, SDS, TBE, trypsin/EDTA, Lipofectamine®, PLUSTM reagent, Superscript® III first-strand synthesis system for RT-PCR from Thermo Fisher Scientific (Waltham, MA); Triton X-114 from Acros Organics (Morris, NJ); Z-IETD from R & D Systems (Minneapolis, MN); Fas ligand from GeneTex (Irvine, CA); anti-CERS6, anti-FLIP and anti-GAPDH antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fas and anti-FADD from BD Transduction Laboratories (San Jose, CA); anti-HA antibody from Roche (Indianapolis, IN); anti-caspase-3, anti-cleaved-caspase-3, anti-caspase-8 and anti-PARP from Cell Signaling Technology (Danvers, MA); Age 1-HF, Bam H1-HF, Eco R1-HF, Mlu 1-HF, Pme 1 and Sgf 1 restriction enzymes from New England Biolabs (Ipswich, MA); bovine serum albumin from Jackson ImmunoResearch Laboratories (West Grove, PA); all oligos were synthesized from Integrated DNA Technologies (Coralville, IA).

    Techniques: Over Expression, Transduction, Plasmid Preparation, High Performance Liquid Chromatography, Mass Spectrometry, Concentration Assay, Apoptosis Assay, Flow Cytometry, Cytometry, Positive Control

    Cyclin B1-Cdc2 complex maintains G2/M arrest (A) Cell cycle analysis of IOMM Lee, CH 157 MN and SF3061 cells treated with 10 μM inhibitor for 1 hr and/or 7 Gy radiation, stained with propidium iodide, and measured 24 hrs after treatment. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells out of 10,000 events were calculated without gating. (B) IOMM Lee, CH 157 MN and SF3061 cells (1×10 5 ) were seeded in 6-well plates and irradiated. Cells were trypsinized and seeded at 1×10 4 cells per well in 96-well plates. After the indicated hours of incubation, MTT reagent was added, followed by another 4 hrs of incubation and the addition of acid-isopropanol. Absorbance was measured at 550 nm and the values were quantified. (C) Western blot analysis of pChk2, cyclin B1 and pCdc2 in the inhibitor-treated cells. GAPDH served as a loading control. Each blot is representative of three independent experiments.

    Journal: Cancer letters

    Article Title: Chk2-mediated G2/M Cell Cycle Arrest Maintains Radiation Resistance in Malignant Meningioma Cells

    doi: 10.1016/j.canlet.2011.08.022

    Figure Lengend Snippet: Cyclin B1-Cdc2 complex maintains G2/M arrest (A) Cell cycle analysis of IOMM Lee, CH 157 MN and SF3061 cells treated with 10 μM inhibitor for 1 hr and/or 7 Gy radiation, stained with propidium iodide, and measured 24 hrs after treatment. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells out of 10,000 events were calculated without gating. (B) IOMM Lee, CH 157 MN and SF3061 cells (1×10 5 ) were seeded in 6-well plates and irradiated. Cells were trypsinized and seeded at 1×10 4 cells per well in 96-well plates. After the indicated hours of incubation, MTT reagent was added, followed by another 4 hrs of incubation and the addition of acid-isopropanol. Absorbance was measured at 550 nm and the values were quantified. (C) Western blot analysis of pChk2, cyclin B1 and pCdc2 in the inhibitor-treated cells. GAPDH served as a loading control. Each blot is representative of three independent experiments.

    Article Snippet: Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C, treated with NSC 109555 ditosylate or Nutlin or Aloisine (Santa Cruz Biotechnology, Santa Cruz, CA), and incubated for the indicated period of time in complete medium. pChk2 (Thr 68), Chk2, p53, cyclin B1, Cdc25C, pCdc25c (Ser 216), pCdc2 (Thr 14/Tyr15) and GAPDH antibodies were obtained from (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Cell Cycle Assay, Staining, Cell Counting, Irradiation, Incubation, MTT Assay, Western Blot

    Irradiated meningioma form aggressive tumors while uPAR knock down induces sustained G2/M arrest in vivo (A-B) IOMM Lee and CH 157 MN luciferase expressing stable cells (1×10 5 ) were implanted into nude mice (4 to 6 weeks old). The first group was treated with irradiated (7 Gy) cells. The second group was infused with irradiated (7 Gy) cells that were allowed to recover. The third group was infused with non-irradiated cells. Tumor progression and morphological and behavioral patterns were followed daily for two weeks with an in vivo imaging system. (C) Representative photmicrograph (40X)of Immunohistochemistry for Cyclin B1 in the brain sections of animals implanted with transfected IOMM Lee cells in combination with radiation treatment (IR; 7Gy) as indicated (n=5). (D) H E staining of brain sections for the visualization of tumor formation. (E) Representative image of RT-PCR analysis for Cyclin B1 in the brain tissues of different treatment groups. GAPDH served as loading control (n=3).

    Journal: Cancer letters

    Article Title: Chk2-mediated G2/M Cell Cycle Arrest Maintains Radiation Resistance in Malignant Meningioma Cells

    doi: 10.1016/j.canlet.2011.08.022

    Figure Lengend Snippet: Irradiated meningioma form aggressive tumors while uPAR knock down induces sustained G2/M arrest in vivo (A-B) IOMM Lee and CH 157 MN luciferase expressing stable cells (1×10 5 ) were implanted into nude mice (4 to 6 weeks old). The first group was treated with irradiated (7 Gy) cells. The second group was infused with irradiated (7 Gy) cells that were allowed to recover. The third group was infused with non-irradiated cells. Tumor progression and morphological and behavioral patterns were followed daily for two weeks with an in vivo imaging system. (C) Representative photmicrograph (40X)of Immunohistochemistry for Cyclin B1 in the brain sections of animals implanted with transfected IOMM Lee cells in combination with radiation treatment (IR; 7Gy) as indicated (n=5). (D) H E staining of brain sections for the visualization of tumor formation. (E) Representative image of RT-PCR analysis for Cyclin B1 in the brain tissues of different treatment groups. GAPDH served as loading control (n=3).

    Article Snippet: Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C, treated with NSC 109555 ditosylate or Nutlin or Aloisine (Santa Cruz Biotechnology, Santa Cruz, CA), and incubated for the indicated period of time in complete medium. pChk2 (Thr 68), Chk2, p53, cyclin B1, Cdc25C, pCdc25c (Ser 216), pCdc2 (Thr 14/Tyr15) and GAPDH antibodies were obtained from (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Irradiation, In Vivo, Luciferase, Expressing, Mouse Assay, In Vivo Imaging, Immunohistochemistry, Transfection, Staining, Reverse Transcription Polymerase Chain Reaction

    Expression of cell cycle-related proteins in irradiated cells (A) Western blot analysis of Chk2, phospho-Chk2, p53, Cdc25C, phospho-Cdc25C (Ser216), cyclin B1 and phospho-Cdc2 (Thr 14/Tyr15) in IOMM Lee, CH 157 MN and SF3061 cells at 24 hrs post-irradiation. GAPDH served as a loading control. (B) ImageJ quantification of the molecules in arbitrary units; each value is representative of three independent experiments. (C) Western blot analysis of cyclin B1 and pCdc2 (Thr 14/Tyr 15) at 8 and 48 hrs post-irradiation. GAPDH served as a loading control.

    Journal: Cancer letters

    Article Title: Chk2-mediated G2/M Cell Cycle Arrest Maintains Radiation Resistance in Malignant Meningioma Cells

    doi: 10.1016/j.canlet.2011.08.022

    Figure Lengend Snippet: Expression of cell cycle-related proteins in irradiated cells (A) Western blot analysis of Chk2, phospho-Chk2, p53, Cdc25C, phospho-Cdc25C (Ser216), cyclin B1 and phospho-Cdc2 (Thr 14/Tyr15) in IOMM Lee, CH 157 MN and SF3061 cells at 24 hrs post-irradiation. GAPDH served as a loading control. (B) ImageJ quantification of the molecules in arbitrary units; each value is representative of three independent experiments. (C) Western blot analysis of cyclin B1 and pCdc2 (Thr 14/Tyr 15) at 8 and 48 hrs post-irradiation. GAPDH served as a loading control.

    Article Snippet: Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C, treated with NSC 109555 ditosylate or Nutlin or Aloisine (Santa Cruz Biotechnology, Santa Cruz, CA), and incubated for the indicated period of time in complete medium. pChk2 (Thr 68), Chk2, p53, cyclin B1, Cdc25C, pCdc25c (Ser 216), pCdc2 (Thr 14/Tyr15) and GAPDH antibodies were obtained from (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing, Irradiation, Western Blot

    p53 acts as downstream effector to Chk2 in IOMM Lee cells (A) Cell cycle analysis of IOMM Lee, CH 157 MN and SF3061 cells treated with 10 μM inhibitor for 1 hr and/or 7 Gy radiation, stained with propidium iodide, and measured 24 hrs after treatment. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells out of 10,000 events were calculated without gating. (B) IOMM Lee, CH 157 MN and SF3061 cells (1×10 5 ) were seeded in 6-well plates and irradiated. Next, cells were trypsinized and seeded at 1×10 4 cells per well in 96-well plates. After the indicated hours of incubation, MTT reagent was added, followed by another 4 hrs of incubation and the addition of acid-isopropanol. Absorbance was measured at 550 nm and the values were quantified. (C) Western blot analysis of pChk2, cyclin B1 and pCdc2 in the inhibitor-treated cells. GAPDH served as a loading control. Each blot is a representative of three independent experiments. (D) ImageJ quantification of cyclin B1 and pCdc2 in the inhibitor-treated cells.

    Journal: Cancer letters

    Article Title: Chk2-mediated G2/M Cell Cycle Arrest Maintains Radiation Resistance in Malignant Meningioma Cells

    doi: 10.1016/j.canlet.2011.08.022

    Figure Lengend Snippet: p53 acts as downstream effector to Chk2 in IOMM Lee cells (A) Cell cycle analysis of IOMM Lee, CH 157 MN and SF3061 cells treated with 10 μM inhibitor for 1 hr and/or 7 Gy radiation, stained with propidium iodide, and measured 24 hrs after treatment. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells out of 10,000 events were calculated without gating. (B) IOMM Lee, CH 157 MN and SF3061 cells (1×10 5 ) were seeded in 6-well plates and irradiated. Next, cells were trypsinized and seeded at 1×10 4 cells per well in 96-well plates. After the indicated hours of incubation, MTT reagent was added, followed by another 4 hrs of incubation and the addition of acid-isopropanol. Absorbance was measured at 550 nm and the values were quantified. (C) Western blot analysis of pChk2, cyclin B1 and pCdc2 in the inhibitor-treated cells. GAPDH served as a loading control. Each blot is a representative of three independent experiments. (D) ImageJ quantification of cyclin B1 and pCdc2 in the inhibitor-treated cells.

    Article Snippet: Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C, treated with NSC 109555 ditosylate or Nutlin or Aloisine (Santa Cruz Biotechnology, Santa Cruz, CA), and incubated for the indicated period of time in complete medium. pChk2 (Thr 68), Chk2, p53, cyclin B1, Cdc25C, pCdc25c (Ser 216), pCdc2 (Thr 14/Tyr15) and GAPDH antibodies were obtained from (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Cell Cycle Assay, Staining, Cell Counting, Irradiation, Incubation, MTT Assay, Western Blot

    Meningioma cells recover from radiation induced cell cycle arrest and uPAR knock down induces cell death (A) Cell cycle analysis of IOMM Lee and CH 157 MN cells treated with 7 Gy radiation, stained with propidium iodide, and measured 72 hrs after treatment. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells out of 10,000 events were calculated without gating. (B) Quantification of distribution of cells in the G0/G1 and G2/M phases of the cell cycle. Data presented are means ± SEM calculated from three independent experiments. (C) Western blot analysis of pChk2, cyclin B1 and pCdc2 at 72 hrs after irradiation. GAPDH served as a loading control. Each blot is representative of three independent experiments. (D) Cell cycle analysis of IOMM Lee and CH 157 MN cells transfected with Scrambled vector (SV), ShuPAR, irradiated (7Gy) stained with propidium iodide after 24 hrs treatment as indicated. Percentage of cells in SubG0/G1 population in each treatment group is plotted. Data presented are means ± SEM calculated from three independent experiments. E) IOMM Lee and CH 157 MN cells were transfected either with SV or ShuPAR expressing plasmids alone or in combination with radiation. After 6h radiation treatment cells were transferred into 96 well plates and MTT assay was performed at indicated period of times. Data presented are means ± SEM calculated from three independent experiments. F) Western blot analysis in transfected cells for cyclin B1 and pCdc2 at 24 hrs after irradiation. GAPDH served as a loading control. Each blot is representative of three independent experiments.

    Journal: Cancer letters

    Article Title: Chk2-mediated G2/M Cell Cycle Arrest Maintains Radiation Resistance in Malignant Meningioma Cells

    doi: 10.1016/j.canlet.2011.08.022

    Figure Lengend Snippet: Meningioma cells recover from radiation induced cell cycle arrest and uPAR knock down induces cell death (A) Cell cycle analysis of IOMM Lee and CH 157 MN cells treated with 7 Gy radiation, stained with propidium iodide, and measured 72 hrs after treatment. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells out of 10,000 events were calculated without gating. (B) Quantification of distribution of cells in the G0/G1 and G2/M phases of the cell cycle. Data presented are means ± SEM calculated from three independent experiments. (C) Western blot analysis of pChk2, cyclin B1 and pCdc2 at 72 hrs after irradiation. GAPDH served as a loading control. Each blot is representative of three independent experiments. (D) Cell cycle analysis of IOMM Lee and CH 157 MN cells transfected with Scrambled vector (SV), ShuPAR, irradiated (7Gy) stained with propidium iodide after 24 hrs treatment as indicated. Percentage of cells in SubG0/G1 population in each treatment group is plotted. Data presented are means ± SEM calculated from three independent experiments. E) IOMM Lee and CH 157 MN cells were transfected either with SV or ShuPAR expressing plasmids alone or in combination with radiation. After 6h radiation treatment cells were transferred into 96 well plates and MTT assay was performed at indicated period of times. Data presented are means ± SEM calculated from three independent experiments. F) Western blot analysis in transfected cells for cyclin B1 and pCdc2 at 24 hrs after irradiation. GAPDH served as a loading control. Each blot is representative of three independent experiments.

    Article Snippet: Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C, treated with NSC 109555 ditosylate or Nutlin or Aloisine (Santa Cruz Biotechnology, Santa Cruz, CA), and incubated for the indicated period of time in complete medium. pChk2 (Thr 68), Chk2, p53, cyclin B1, Cdc25C, pCdc25c (Ser 216), pCdc2 (Thr 14/Tyr15) and GAPDH antibodies were obtained from (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Cell Cycle Assay, Staining, Cell Counting, Western Blot, Irradiation, Transfection, Plasmid Preparation, Expressing, MTT Assay

    TCR signaling induces RORγt phosphorylation and subsequent AhR-RORγt interaction. ( A ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, p-IKKβ (Ser 180/181 ), and IKKβ in primary splenic T cells. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( B ) Coimmunoprecipitation of endogenous AhR with RORγt from lysates of murine primary splenic T cells stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( C ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, and IKKβ in primary splenic T cells of IKKβ f/f or CD4-Cre;IKKβ f/f mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( D ) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt (left) or between AhR and Ser 489 -phosphorylated RORγt (right) in primary T cells of IKKβ f/f or IKKβ f/f ;CD4-Cre mice. T cells were stimulated as in (C). Each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Original magnification, ×630; scale bars, 10 μm. ( E and F ) ELISA of various cytokines in supernatants of primary splenic T cells from IKKβ f/f or IKKβ f/f ;CD4-Cre mice (E), as well as RORγt f/f or RORγt f/f ;CD4-Cre mice (F). T cells were stimulated with plate-bound anti-CD3 antibodies (2 μg each per milliliter) for 3 days. Means ± SD are shown. n = 3 per group. ( G ) Immunoblotting of RORγt and GAPDH proteins from primary splenic T cells of RORγt f/f or RORγt f/f ;CD4-Cre mice. Data shown (A to G) are representative of three independent experiments. ( H ) Schematic model of IL-17A transcription induced by the AhR-RORγt complex in GLK-overexpressing or TCR-stimulated T cells. GLK overexpression in T cells of T cell–specific GLK Tg (Lck-GLK Tg) mice induces AhR Ser 36 phosphorylation through PKCθ and also induces RORγt Ser 489 phosphorylation through IKKβ. Once RORγt is phosphorylated, RORγt interacts directly with AhR. Phosphorylated AhR is responsible for transporting RORγt into cell nucleus. The AhR-RORγt complex binds to both the RORγt-binding element (−877 to −872) and the AhR-binding element (−254 to −249) of the IL-17A promoter, leading to induction of IL-17A transcription. In normal T cells, TCR stimulation also induces GLK kinase activity and downstream signaling, including IKKβ activation, RORγt Ser 489 phosphorylation, and the AhR-RORγt interaction. Besides NF-κB, other critical transcription factors [such as nuclear factor of activated T cell 1 (NFAT1) or activator protein 1 (AP-1)] are also required for the transcriptional activation of IL-2, IFN-γ, IL-4, IL-6, and TNF-α in T cells. “Others” denotes other critical transcription factors (table S1). NF-κB is required for TCR-induced production of multiple cytokines; however, the GLK–IKKβ–NF-κB cascade alone is not sufficient for the induction of multiple cytokines. Collectively, GLK overexpression or TCR signaling induces IL-17A transcription through AhR and RORγt in T cells.

    Journal: Science Advances

    Article Title: GLK-IKKβ signaling induces dimerization and translocation of the AhR-RORγt complex in IL-17A induction and autoimmune disease

    doi: 10.1126/sciadv.aat5401

    Figure Lengend Snippet: TCR signaling induces RORγt phosphorylation and subsequent AhR-RORγt interaction. ( A ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, p-IKKβ (Ser 180/181 ), and IKKβ in primary splenic T cells. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( B ) Coimmunoprecipitation of endogenous AhR with RORγt from lysates of murine primary splenic T cells stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( C ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, and IKKβ in primary splenic T cells of IKKβ f/f or CD4-Cre;IKKβ f/f mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( D ) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt (left) or between AhR and Ser 489 -phosphorylated RORγt (right) in primary T cells of IKKβ f/f or IKKβ f/f ;CD4-Cre mice. T cells were stimulated as in (C). Each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Original magnification, ×630; scale bars, 10 μm. ( E and F ) ELISA of various cytokines in supernatants of primary splenic T cells from IKKβ f/f or IKKβ f/f ;CD4-Cre mice (E), as well as RORγt f/f or RORγt f/f ;CD4-Cre mice (F). T cells were stimulated with plate-bound anti-CD3 antibodies (2 μg each per milliliter) for 3 days. Means ± SD are shown. n = 3 per group. ( G ) Immunoblotting of RORγt and GAPDH proteins from primary splenic T cells of RORγt f/f or RORγt f/f ;CD4-Cre mice. Data shown (A to G) are representative of three independent experiments. ( H ) Schematic model of IL-17A transcription induced by the AhR-RORγt complex in GLK-overexpressing or TCR-stimulated T cells. GLK overexpression in T cells of T cell–specific GLK Tg (Lck-GLK Tg) mice induces AhR Ser 36 phosphorylation through PKCθ and also induces RORγt Ser 489 phosphorylation through IKKβ. Once RORγt is phosphorylated, RORγt interacts directly with AhR. Phosphorylated AhR is responsible for transporting RORγt into cell nucleus. The AhR-RORγt complex binds to both the RORγt-binding element (−877 to −872) and the AhR-binding element (−254 to −249) of the IL-17A promoter, leading to induction of IL-17A transcription. In normal T cells, TCR stimulation also induces GLK kinase activity and downstream signaling, including IKKβ activation, RORγt Ser 489 phosphorylation, and the AhR-RORγt interaction. Besides NF-κB, other critical transcription factors [such as nuclear factor of activated T cell 1 (NFAT1) or activator protein 1 (AP-1)] are also required for the transcriptional activation of IL-2, IFN-γ, IL-4, IL-6, and TNF-α in T cells. “Others” denotes other critical transcription factors (table S1). NF-κB is required for TCR-induced production of multiple cytokines; however, the GLK–IKKβ–NF-κB cascade alone is not sufficient for the induction of multiple cytokines. Collectively, GLK overexpression or TCR signaling induces IL-17A transcription through AhR and RORγt in T cells.

    Article Snippet: Anti-AhR (clone RPT9), anti–p-IKKβ (Ser180/181 ; #ab55341), and anti-GAPDH (clone mAbcam 9484, catalog #ab9482) antibodies were purchased from Abcam.

    Techniques: Mouse Assay, Confocal Microscopy, Staining, Enzyme-linked Immunosorbent Assay, Over Expression, Binding Assay, Activity Assay, Activation Assay

    Wnt5a expressing tumors have less Wnt/β-catenin signaling than MMTV-Wnt1 tumors. (A) Quantitative RT-PCR of Wnt/ β -catenin target genes . Expression of Axin2 mRNA in MMTV-Wnt1 versus MMTV-Wnt1;MMTV-Wnt5a tumors as determined by quantitative RT-PCR (n = 5 MMTV-Wnt1, n = 5 MMTV-Wnt1;MMTV-Wnt5a). Data are shown as tables obtained using REST software. Axin2 mRNA was significantly down-regulated in MMTV-Wnt1;MMTV-Wnt5a tumors. (B) Western blot for β-catenin protein . Protein lysates were prepared from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a tumors. β-catenin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls. The ratio of active β-catenin to β-catenin as determined by densitometic analysis is shown. MMTV-Wnt1;MMTV-Wnt5a tumors displayed decreased levels of active β-catenin compared to controls.

    Journal: PLoS ONE

    Article Title: Wnt5a Suppresses Tumor Formation and Redirects Tumor Phenotype in MMTV-Wnt1 Tumors

    doi: 10.1371/journal.pone.0113247

    Figure Lengend Snippet: Wnt5a expressing tumors have less Wnt/β-catenin signaling than MMTV-Wnt1 tumors. (A) Quantitative RT-PCR of Wnt/ β -catenin target genes . Expression of Axin2 mRNA in MMTV-Wnt1 versus MMTV-Wnt1;MMTV-Wnt5a tumors as determined by quantitative RT-PCR (n = 5 MMTV-Wnt1, n = 5 MMTV-Wnt1;MMTV-Wnt5a). Data are shown as tables obtained using REST software. Axin2 mRNA was significantly down-regulated in MMTV-Wnt1;MMTV-Wnt5a tumors. (B) Western blot for β-catenin protein . Protein lysates were prepared from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a tumors. β-catenin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls. The ratio of active β-catenin to β-catenin as determined by densitometic analysis is shown. MMTV-Wnt1;MMTV-Wnt5a tumors displayed decreased levels of active β-catenin compared to controls.

    Article Snippet: To ensure equal protein loading between lanes, the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Santa Cruz) was determined for each blot.

    Techniques: Expressing, Quantitative RT-PCR, Software, Western Blot

    Wnt5a expressing tumors demonstrate a decrease in markers of the basal tumor subtype. (A) Western blot using protein lysates isolated from the epithelium of MMTV-Wnt1 and MMTV-1;MMTV-Wnt5a mammary tumors. The expression of molecular markers of basal and luminal tumor subtypes were compared. Keratin 6 and Keratin 5 were strongly down-regulated in Wnt5a expressing tumors. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B–D) Immunostaining for K6 . Sections from MMTV-Wnt1 (B) and MMTV-1;MMTV-Wnt5a (C) tumors were stained with anti-Keratin 6 antibody using immunofluorescence (K6 = green; nuclei = blue). The percentage of cells expressing K6 was determined and graphed (D). Values are means +/− standard error (n = 6 MMTV-Wnt1, 3 fields per tumor; n = 5 MMTV-Wnt1;MMTV-Wnt5a, 3 fields per tumor). MMTV-Wnt1;MMTV-Wnt5a tumors demonstrated a significant decrease in K6-expressing cells as measured by T-test (* = p

    Journal: PLoS ONE

    Article Title: Wnt5a Suppresses Tumor Formation and Redirects Tumor Phenotype in MMTV-Wnt1 Tumors

    doi: 10.1371/journal.pone.0113247

    Figure Lengend Snippet: Wnt5a expressing tumors demonstrate a decrease in markers of the basal tumor subtype. (A) Western blot using protein lysates isolated from the epithelium of MMTV-Wnt1 and MMTV-1;MMTV-Wnt5a mammary tumors. The expression of molecular markers of basal and luminal tumor subtypes were compared. Keratin 6 and Keratin 5 were strongly down-regulated in Wnt5a expressing tumors. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B–D) Immunostaining for K6 . Sections from MMTV-Wnt1 (B) and MMTV-1;MMTV-Wnt5a (C) tumors were stained with anti-Keratin 6 antibody using immunofluorescence (K6 = green; nuclei = blue). The percentage of cells expressing K6 was determined and graphed (D). Values are means +/− standard error (n = 6 MMTV-Wnt1, 3 fields per tumor; n = 5 MMTV-Wnt1;MMTV-Wnt5a, 3 fields per tumor). MMTV-Wnt1;MMTV-Wnt5a tumors demonstrated a significant decrease in K6-expressing cells as measured by T-test (* = p

    Article Snippet: To ensure equal protein loading between lanes, the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Santa Cruz) was determined for each blot.

    Techniques: Expressing, Western Blot, Isolation, Immunostaining, Staining, Immunofluorescence

    Ectopic expression of Wnt5a results in low expression of K6 and K14. Expression of molecular markers for basal and luminal progenitors in MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a mammary glands was compared by western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Each lane contains protein isolated from a separate mouse.

    Journal: PLoS ONE

    Article Title: Wnt5a Suppresses Tumor Formation and Redirects Tumor Phenotype in MMTV-Wnt1 Tumors

    doi: 10.1371/journal.pone.0113247

    Figure Lengend Snippet: Ectopic expression of Wnt5a results in low expression of K6 and K14. Expression of molecular markers for basal and luminal progenitors in MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a mammary glands was compared by western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Each lane contains protein isolated from a separate mouse.

    Article Snippet: To ensure equal protein loading between lanes, the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Santa Cruz) was determined for each blot.

    Techniques: Expressing, Western Blot, Isolation

    Wnt/β-catenin signaling is not down-regulated in Wnt5a expressing MMTV-Wnt1 glands. (A) Western blot analysis of β-catenin protein in MMTV-Wnt1mammary gland . The level of active β-catenin was compared in protein lysates from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a mammary glands. Total β-catenin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls. The ratio of active β-catenin-to-β-catenin is shown at the bottom on the gel. Each lane represents a sample from a different mouse. (B) Quantitative RT-PCR of Axin2, a Wnt/β-catenin target gene, in unsorted primary mammary epithelial cells . Expression of Axin2 and hWnt5a m RNA in unsorted PMECs from MMTV-Wnt1;MMTV-Wnt5a vs. MMTV-Wnt1 mammary glands was determined by quantitative RT-PCR (n = 12 MMTV-Wnt1, n = 11 MMTV-Wnt1;MMTV-Wnt5a separate mice). Data are shown as tables obtained using REST analysis software. Expression = fold difference in MMTV-Wnt1;Wnt5a relative to MMTV-Wnt1 controls after normalization to Gapdh. (C) Quantitative RT-PCR of Axin2 in E-cadherin negative primary mammary epithelial cells . Expression of Axin2 and hWnt5a mRNA in E-cadherin (Ecad) negative PMECs from MMTV-Wnt1;MMTV-Wnt5a vs. MMTV-Wnt1 mammary glands was determined by quantitative RT-PCR (n = 2 MMTV-Wnt1, n = 2 MMTV-Wnt1;MMTV-Wnt5a separate mice). Data are shown as tables obtained using REST analysis software. Expression = fold difference in MMTV-Wnt1;Wnt5a relative to MMTV-Wnt1 controls after normalization to Gapdh.

    Journal: PLoS ONE

    Article Title: Wnt5a Suppresses Tumor Formation and Redirects Tumor Phenotype in MMTV-Wnt1 Tumors

    doi: 10.1371/journal.pone.0113247

    Figure Lengend Snippet: Wnt/β-catenin signaling is not down-regulated in Wnt5a expressing MMTV-Wnt1 glands. (A) Western blot analysis of β-catenin protein in MMTV-Wnt1mammary gland . The level of active β-catenin was compared in protein lysates from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a mammary glands. Total β-catenin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls. The ratio of active β-catenin-to-β-catenin is shown at the bottom on the gel. Each lane represents a sample from a different mouse. (B) Quantitative RT-PCR of Axin2, a Wnt/β-catenin target gene, in unsorted primary mammary epithelial cells . Expression of Axin2 and hWnt5a m RNA in unsorted PMECs from MMTV-Wnt1;MMTV-Wnt5a vs. MMTV-Wnt1 mammary glands was determined by quantitative RT-PCR (n = 12 MMTV-Wnt1, n = 11 MMTV-Wnt1;MMTV-Wnt5a separate mice). Data are shown as tables obtained using REST analysis software. Expression = fold difference in MMTV-Wnt1;Wnt5a relative to MMTV-Wnt1 controls after normalization to Gapdh. (C) Quantitative RT-PCR of Axin2 in E-cadherin negative primary mammary epithelial cells . Expression of Axin2 and hWnt5a mRNA in E-cadherin (Ecad) negative PMECs from MMTV-Wnt1;MMTV-Wnt5a vs. MMTV-Wnt1 mammary glands was determined by quantitative RT-PCR (n = 2 MMTV-Wnt1, n = 2 MMTV-Wnt1;MMTV-Wnt5a separate mice). Data are shown as tables obtained using REST analysis software. Expression = fold difference in MMTV-Wnt1;Wnt5a relative to MMTV-Wnt1 controls after normalization to Gapdh.

    Article Snippet: To ensure equal protein loading between lanes, the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Santa Cruz) was determined for each blot.

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Mouse Assay, Software