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  • 99
    Thermo Fisher gene exp gapdh hs99999905 m1
    FAK activity of HMEC-1 residing on soft PA hydrogels is decreased, as is Lm uptake. (A) Western blots from whole HMEC-1 lysates showing expression of phosphorylated FAK (Tyr397) and total FAK for cells residing on soft gels (3 kPa), stiff gels (70 kPa), and TC polystyrene substrates with or without 2 μM PF537228 FAK inhibitor. In each Western blot, equal quantities of protein were loaded and equal loading was confirmed in relation to glyceraldehyde 3-phosphate dehydrogenase <t>(GAPDH)</t> expression. In each case, the Western blots shown are representative of three independent experiments. (B, C) Normalized ratios of FAK/GAPDH (B) and pFAK (Tyr397)/GAPDH (C) for HMEC-1 residing on varying-stiffness substrates and treated or not with 2 μM PF537228 FAK inhibitor. Different color circles correspond to data from three independent experiments. Black bars represent the means of the three independent experiments. For each experiment, values have been normalized relative to the ratio for cells residing on polystyrene substrates. (D) Inhibition of bacterial uptake by FAK inhibitors. FAK-14, PF573228, or vehicle control was added 1 h before addition of bacteria to HMEC-1 residing on polystyrene substrates. Percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of inhibitor concentration (mean ± SD, N = 4 replicates). x = 0 corresponds to cells treated with vehicle control. Inset shows the same data with concentration on a log scale. Infection was analyzed by flow cytometry, 7–8 h after infection. MOI is 80. Representative data come from one of three independent experiments. (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or FAK siRNA (siFAK) (means ± SD, three independent experiments and N = 6 replicates per experiment). MOI is 60 (gray) or 20 (green). Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (
    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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    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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    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 18820 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc 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 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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    Thermo Fisher gene exp gapdh mm99999915 g1
    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
    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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    99
    Thermo Fisher gapdh
    K63-linked ubiquitination of YAP induces its growth-promoting function. a Left panel: immunoblotting of YAP and <t>GAPDH</t> in HMLE cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). Right panel: growth curves. n = 5 biological replicates. b Images (upper panel) and quantification (lower panel) of soft agar colony formation by the cells described in ( a ). n = 3 biological replicates. c Endpoint images of tumors dissected from NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. d , e Tumor growth curves ( d ) and tumor weight (at the endpoint, e ) of NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. f Left panel: immunoblotting of YAP, <t>SKP2,</t> and GAPDH in BT549 cells transduced with MYC-SKP2 with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. g Left panel: immunoblotting of YAP, OTUD1 and GAPDH in BT549 cells transduced with OTUD1 shRNA with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. h , i Kaplan–Meier curves of recurrence-free survival of patients with breast cancer, stratified by SKP2 ( n = 3951 patients, probe: 203625_x_at, ( h ) or OTUD1 ( n = 1764 patients, probe: 226140_s_at, ( i . Auto-select best cutoff was used in the analysis. j Model for the regulation of YAP localization and activity by non-proteolytic ubiquitination. Error bars in a , b , and d – g are s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. * P
    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
    K63-linked ubiquitination of YAP induces its growth-promoting function. a Left panel: immunoblotting of YAP and <t>GAPDH</t> in HMLE cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). Right panel: growth curves. n = 5 biological replicates. b Images (upper panel) and quantification (lower panel) of soft agar colony formation by the cells described in ( a ). n = 3 biological replicates. c Endpoint images of tumors dissected from NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. d , e Tumor growth curves ( d ) and tumor weight (at the endpoint, e ) of NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. f Left panel: immunoblotting of YAP, <t>SKP2,</t> and GAPDH in BT549 cells transduced with MYC-SKP2 with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. g Left panel: immunoblotting of YAP, OTUD1 and GAPDH in BT549 cells transduced with OTUD1 shRNA with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. h , i Kaplan–Meier curves of recurrence-free survival of patients with breast cancer, stratified by SKP2 ( n = 3951 patients, probe: 203625_x_at, ( h ) or OTUD1 ( n = 1764 patients, probe: 226140_s_at, ( i . Auto-select best cutoff was used in the analysis. j Model for the regulation of YAP localization and activity by non-proteolytic ubiquitination. Error bars in a , b , and d – g are s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. * P
    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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    99
    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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    99
    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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    94
    Abcam anti gapdh antibody 6c5
    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 6c5, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 1000 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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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    99
    Cell Signaling Technology Inc 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.
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    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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    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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    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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    Proteintech mouse gapdh 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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    Proteintech rabbit gapdh polyclonal
    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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    Thermo Fisher gene exp gapdh hs02786624 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.
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    Thermo Fisher gene exp gapdh rn01775763 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.
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    Thermo Fisher human gapd gapdh endogenous control
    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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    Thermo Fisher gene exp gapdh hs03929097 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 Hs03929097 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1452 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    This gene encodes a protein belonging to the glyceraldehyde 3 phosphate dehydrogenase family of enzymes that play an important role in carbohydrate metabolism Like its somatic cell counterpart this sperm
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    Mouse monoclonal GAPDHS antibody
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    Image Search Results


    FAK activity of HMEC-1 residing on soft PA hydrogels is decreased, as is Lm uptake. (A) Western blots from whole HMEC-1 lysates showing expression of phosphorylated FAK (Tyr397) and total FAK for cells residing on soft gels (3 kPa), stiff gels (70 kPa), and TC polystyrene substrates with or without 2 μM PF537228 FAK inhibitor. In each Western blot, equal quantities of protein were loaded and equal loading was confirmed in relation to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. In each case, the Western blots shown are representative of three independent experiments. (B, C) Normalized ratios of FAK/GAPDH (B) and pFAK (Tyr397)/GAPDH (C) for HMEC-1 residing on varying-stiffness substrates and treated or not with 2 μM PF537228 FAK inhibitor. Different color circles correspond to data from three independent experiments. Black bars represent the means of the three independent experiments. For each experiment, values have been normalized relative to the ratio for cells residing on polystyrene substrates. (D) Inhibition of bacterial uptake by FAK inhibitors. FAK-14, PF573228, or vehicle control was added 1 h before addition of bacteria to HMEC-1 residing on polystyrene substrates. Percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of inhibitor concentration (mean ± SD, N = 4 replicates). x = 0 corresponds to cells treated with vehicle control. Inset shows the same data with concentration on a log scale. Infection was analyzed by flow cytometry, 7–8 h after infection. MOI is 80. Representative data come from one of three independent experiments. (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or FAK siRNA (siFAK) (means ± SD, three independent experiments and N = 6 replicates per experiment). MOI is 60 (gray) or 20 (green). Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (

    Journal: Molecular Biology of the Cell

    Article Title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

    doi: 10.1091/mbc.E18-04-0228

    Figure Lengend Snippet: FAK activity of HMEC-1 residing on soft PA hydrogels is decreased, as is Lm uptake. (A) Western blots from whole HMEC-1 lysates showing expression of phosphorylated FAK (Tyr397) and total FAK for cells residing on soft gels (3 kPa), stiff gels (70 kPa), and TC polystyrene substrates with or without 2 μM PF537228 FAK inhibitor. In each Western blot, equal quantities of protein were loaded and equal loading was confirmed in relation to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. In each case, the Western blots shown are representative of three independent experiments. (B, C) Normalized ratios of FAK/GAPDH (B) and pFAK (Tyr397)/GAPDH (C) for HMEC-1 residing on varying-stiffness substrates and treated or not with 2 μM PF537228 FAK inhibitor. Different color circles correspond to data from three independent experiments. Black bars represent the means of the three independent experiments. For each experiment, values have been normalized relative to the ratio for cells residing on polystyrene substrates. (D) Inhibition of bacterial uptake by FAK inhibitors. FAK-14, PF573228, or vehicle control was added 1 h before addition of bacteria to HMEC-1 residing on polystyrene substrates. Percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of inhibitor concentration (mean ± SD, N = 4 replicates). x = 0 corresponds to cells treated with vehicle control. Inset shows the same data with concentration on a log scale. Infection was analyzed by flow cytometry, 7–8 h after infection. MOI is 80. Representative data come from one of three independent experiments. (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or FAK siRNA (siFAK) (means ± SD, three independent experiments and N = 6 replicates per experiment). MOI is 60 (gray) or 20 (green). Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (

    Article Snippet: Genes of interest were amplified using primers Hs00958113_g1 for Vimentin (ThermoFisher; 4331182) and Hs99999905_m1 for GAPDH (ThermoFisher; 4333764) on a StepOnePlus real-time PCR system.

    Techniques: Activity Assay, Western Blot, Expressing, Inhibition, Infection, Concentration Assay, Flow Cytometry, Cytometry, MANN-WHITNEY

    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

    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

    K63-linked ubiquitination of YAP induces its growth-promoting function. a Left panel: immunoblotting of YAP and GAPDH in HMLE cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). Right panel: growth curves. n = 5 biological replicates. b Images (upper panel) and quantification (lower panel) of soft agar colony formation by the cells described in ( a ). n = 3 biological replicates. c Endpoint images of tumors dissected from NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. d , e Tumor growth curves ( d ) and tumor weight (at the endpoint, e ) of NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. f Left panel: immunoblotting of YAP, SKP2, and GAPDH in BT549 cells transduced with MYC-SKP2 with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. g Left panel: immunoblotting of YAP, OTUD1 and GAPDH in BT549 cells transduced with OTUD1 shRNA with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. h , i Kaplan–Meier curves of recurrence-free survival of patients with breast cancer, stratified by SKP2 ( n = 3951 patients, probe: 203625_x_at, ( h ) or OTUD1 ( n = 1764 patients, probe: 226140_s_at, ( i . Auto-select best cutoff was used in the analysis. j Model for the regulation of YAP localization and activity by non-proteolytic ubiquitination. Error bars in a , b , and d – g are s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. * P

    Journal: Nature Communications

    Article Title: SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity

    doi: 10.1038/s41467-018-04620-y

    Figure Lengend Snippet: K63-linked ubiquitination of YAP induces its growth-promoting function. a Left panel: immunoblotting of YAP and GAPDH in HMLE cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). Right panel: growth curves. n = 5 biological replicates. b Images (upper panel) and quantification (lower panel) of soft agar colony formation by the cells described in ( a ). n = 3 biological replicates. c Endpoint images of tumors dissected from NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. d , e Tumor growth curves ( d ) and tumor weight (at the endpoint, e ) of NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. f Left panel: immunoblotting of YAP, SKP2, and GAPDH in BT549 cells transduced with MYC-SKP2 with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. g Left panel: immunoblotting of YAP, OTUD1 and GAPDH in BT549 cells transduced with OTUD1 shRNA with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. h , i Kaplan–Meier curves of recurrence-free survival of patients with breast cancer, stratified by SKP2 ( n = 3951 patients, probe: 203625_x_at, ( h ) or OTUD1 ( n = 1764 patients, probe: 226140_s_at, ( i . Auto-select best cutoff was used in the analysis. j Model for the regulation of YAP localization and activity by non-proteolytic ubiquitination. Error bars in a , b , and d – g are s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. * P

    Article Snippet: Antibodies against SKP2 (32-3300, 1:500), Xpress (R910-25, 1:1000), and GAPDH (MA5-15738, 1:3000) were from ThermoFisher Scientific.

    Techniques: Transduction, Mutagenesis, Mouse Assay, Injection, Multiple Displacement Amplification, shRNA, Activity Assay, Two Tailed Test

    K63-linked ubiquitination of YAP is independent of Hippo signaling-mediated YAP phosphorylation. a Immunoblotting of p-YAP (S127), FLAG-YAP, and GAPDH in HEK293T cells transfected with SFB-YAP (wild-type, K321R, K497R, or K321R/K497R). b YAP knockout HEK293A cells (generated by CRISPR-Cas9) were transfected with SFB-YAP (S127A or S127A/K321R/K497R) and immunostained with a YAP-specific antibody (green). DAPI (blue) was used to stain DNA. Two different fields are shown. Scale bar, 20 μm. c Immunoblotting of p-YAP (S127), YAP, and SKP2 in HEK293A cells stably expressing MYC-SKP2 (upper panel) or SKP2 shRNA ( lower panel). Scr: a scramble control. d Immunoblotting of p-YAP (S127), YAP, OTUD1, and GAPDH in HEK293A cells stably transfected with HA-OTUD1 (upper panel) or OTUD1 shRNA (lower panel). Scr: a scramble control. e HEK293T cells were co-transfected with SFB-YAP (wild-type or S127A), MYC-SKP2 and the K63-specific mutant of HA-ubiquitin and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against HA and FLAG. f HEK293T cells were co-transfected SFB-YAP (wild-type or S127A), FLAG-OTUD1 and the K63-specific mutant of HA-ubiquitin and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against HA and FLAG. g YAP knockout HEK293A cells (generated by CRISPR-Cas9) were transfected with SFB-YAP (WT or S127A) with or without SKP2 shRNA (on a GFP-positive pGIPZ vector), and immunostained with a YAP-specific antibody (red). DAPI (blue) was used to stain DNA. Scr: a scramble control. Scale bar, 20 μm

    Journal: Nature Communications

    Article Title: SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity

    doi: 10.1038/s41467-018-04620-y

    Figure Lengend Snippet: K63-linked ubiquitination of YAP is independent of Hippo signaling-mediated YAP phosphorylation. a Immunoblotting of p-YAP (S127), FLAG-YAP, and GAPDH in HEK293T cells transfected with SFB-YAP (wild-type, K321R, K497R, or K321R/K497R). b YAP knockout HEK293A cells (generated by CRISPR-Cas9) were transfected with SFB-YAP (S127A or S127A/K321R/K497R) and immunostained with a YAP-specific antibody (green). DAPI (blue) was used to stain DNA. Two different fields are shown. Scale bar, 20 μm. c Immunoblotting of p-YAP (S127), YAP, and SKP2 in HEK293A cells stably expressing MYC-SKP2 (upper panel) or SKP2 shRNA ( lower panel). Scr: a scramble control. d Immunoblotting of p-YAP (S127), YAP, OTUD1, and GAPDH in HEK293A cells stably transfected with HA-OTUD1 (upper panel) or OTUD1 shRNA (lower panel). Scr: a scramble control. e HEK293T cells were co-transfected with SFB-YAP (wild-type or S127A), MYC-SKP2 and the K63-specific mutant of HA-ubiquitin and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against HA and FLAG. f HEK293T cells were co-transfected SFB-YAP (wild-type or S127A), FLAG-OTUD1 and the K63-specific mutant of HA-ubiquitin and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against HA and FLAG. g YAP knockout HEK293A cells (generated by CRISPR-Cas9) were transfected with SFB-YAP (WT or S127A) with or without SKP2 shRNA (on a GFP-positive pGIPZ vector), and immunostained with a YAP-specific antibody (red). DAPI (blue) was used to stain DNA. Scr: a scramble control. Scale bar, 20 μm

    Article Snippet: Antibodies against SKP2 (32-3300, 1:500), Xpress (R910-25, 1:1000), and GAPDH (MA5-15738, 1:3000) were from ThermoFisher Scientific.

    Techniques: Transfection, Knock-Out, Generated, CRISPR, Staining, Stable Transfection, Expressing, shRNA, Mutagenesis, Plasmid Preparation

    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