phospho ck2 substrate  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc phospho ck2 substrate
    Proteins Co-Immunoprecipitating With NOXO1 in T84 Cells Stimulated by TNFα (5 ng/mL) + IL17 (50 ng/mL) and Related to NADPH Oxidase or Redox Signaling
    Phospho Ck2 Substrate, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho ck2 substrate/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho ck2 substrate - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation"

    Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2022.01.003

    Proteins Co-Immunoprecipitating With NOXO1 in T84 Cells Stimulated by TNFα (5 ng/mL) + IL17 (50 ng/mL) and Related to NADPH Oxidase or Redox Signaling
    Figure Legend Snippet: Proteins Co-Immunoprecipitating With NOXO1 in T84 Cells Stimulated by TNFα (5 ng/mL) + IL17 (50 ng/mL) and Related to NADPH Oxidase or Redox Signaling

    Techniques Used: Binding Assay, Protein Binding

    CK2 interacts with NOXO1 in T84 colon epithelial cells under inflammatory conditions. ( A ) Immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon T84 cells stimulated with TNFα (5 ng/mL) or IL17 (50 ng/mL) individually or in combination for 24 hours at 37°C. Representative of 3 independent experiments. ( B ) ROS production was measured by chemiluminescence in T84 cells stimulated as in (A) . n = 3. ( C ) Immunoblots of CK2 α/α′ in NOXO1 IPP from T84 cells stimulated as in (A) . Representative of 4 independent experiments. ( D ) Densitometry analysis of CK2 α/α′ in NOXO1 IPP as in ( C ), normalized to CK2 α/α′expression in cell lysates; n = 4; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05. ( E ) Confocal microcopy of T84 cells co-stimulated or not with TNFα + IL17 for 24 hours at 37°C. NOXO1 ( green ), CK2 α/α′ ( red ), DAPI ( blue ). Scale bar: 10 μm. Representative of 6 independent experiments.
    Figure Legend Snippet: CK2 interacts with NOXO1 in T84 colon epithelial cells under inflammatory conditions. ( A ) Immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon T84 cells stimulated with TNFα (5 ng/mL) or IL17 (50 ng/mL) individually or in combination for 24 hours at 37°C. Representative of 3 independent experiments. ( B ) ROS production was measured by chemiluminescence in T84 cells stimulated as in (A) . n = 3. ( C ) Immunoblots of CK2 α/α′ in NOXO1 IPP from T84 cells stimulated as in (A) . Representative of 4 independent experiments. ( D ) Densitometry analysis of CK2 α/α′ in NOXO1 IPP as in ( C ), normalized to CK2 α/α′expression in cell lysates; n = 4; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05. ( E ) Confocal microcopy of T84 cells co-stimulated or not with TNFα + IL17 for 24 hours at 37°C. NOXO1 ( green ), CK2 α/α′ ( red ), DAPI ( blue ). Scale bar: 10 μm. Representative of 6 independent experiments.

    Techniques Used: Western Blot, Expressing

    CK2 interacts directly with NOXO1 through the N-terminal region mostly containing the PX domain. ( A ) Coomassie Blue staining of recombinant NOXO1, p47 PHOX , NOXA1, and p67 PHOX . ( B ) Dot-blot analysis of interaction between CK2 and recombinant NOXO1, p47 PHOX , NOXA1, or p67 PHOX . Representative of 3 independent experiments. ( C ) Schematic representation and Coomassie Blue staining of GST fusion proteins of the NOXO1 full-length (β isoform), N-terminal (1-157), and C-terminal regions (232-371). ( D ) Pull-down of CK2 from resting T84 cells lysates by full-length GST-NOXO1, GST-NOXO1 (1-157), GST-NOXO1 (232-371), or GST (control). Representative of 3 independent experiments. ( E ) Densitometry analysis of CK2 α/α′ pull-downed as in ( D ), normalized to GST-fusion proteins; n = 3; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗∗∗ P < 0.0001.
    Figure Legend Snippet: CK2 interacts directly with NOXO1 through the N-terminal region mostly containing the PX domain. ( A ) Coomassie Blue staining of recombinant NOXO1, p47 PHOX , NOXA1, and p67 PHOX . ( B ) Dot-blot analysis of interaction between CK2 and recombinant NOXO1, p47 PHOX , NOXA1, or p67 PHOX . Representative of 3 independent experiments. ( C ) Schematic representation and Coomassie Blue staining of GST fusion proteins of the NOXO1 full-length (β isoform), N-terminal (1-157), and C-terminal regions (232-371). ( D ) Pull-down of CK2 from resting T84 cells lysates by full-length GST-NOXO1, GST-NOXO1 (1-157), GST-NOXO1 (232-371), or GST (control). Representative of 3 independent experiments. ( E ) Densitometry analysis of CK2 α/α′ pull-downed as in ( D ), normalized to GST-fusion proteins; n = 3; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗∗∗ P < 0.0001.

    Techniques Used: Staining, Recombinant, Dot Blot

    CK2 phosphorylates recombinant NOXO1 in vitro on several sites. ( A ) Autoradiography of recombinant NOXO1 phosphorylated with constitutively active CK2 in the presence of radioactive [γ -32P ] ATP. Time course ( left ), with or without CK2 ( right ). Representative of 3 independent experiments. ( B ) MS/MS protein coverage of recombinant NOXO1 after in vitro phosphorylation by CK2. Peptides were filtered according to 1% false discovery rate that corresponds to an identification score value >37.6 (–10lgP) with the software Peaks Studio Xpro. The confidence of modification sites is estimated by an Ascore, which calculates an ambiguity score as –10 × log 10 (p). The P value indicates the likelihood that the peptide is matched by chance (Ascore = 20 for P value of .01). Only confident modification sites with Ascore >20 were retained and their position labeled ( red ). ( C ) XIC of the heavily C-terminus phosphorylated peptide of m/z 753.997 showing 3 distinct elution peaks for 3 distinct phosphorylation sites. ( D ) Representative MS/MS spectrum of the phosphorylated C-terminus peptide at position 368. The confidence of modification sites is estimated by an Ascore (Ascore = 20 for P value of .01).
    Figure Legend Snippet: CK2 phosphorylates recombinant NOXO1 in vitro on several sites. ( A ) Autoradiography of recombinant NOXO1 phosphorylated with constitutively active CK2 in the presence of radioactive [γ -32P ] ATP. Time course ( left ), with or without CK2 ( right ). Representative of 3 independent experiments. ( B ) MS/MS protein coverage of recombinant NOXO1 after in vitro phosphorylation by CK2. Peptides were filtered according to 1% false discovery rate that corresponds to an identification score value >37.6 (–10lgP) with the software Peaks Studio Xpro. The confidence of modification sites is estimated by an Ascore, which calculates an ambiguity score as –10 × log 10 (p). The P value indicates the likelihood that the peptide is matched by chance (Ascore = 20 for P value of .01). Only confident modification sites with Ascore >20 were retained and their position labeled ( red ). ( C ) XIC of the heavily C-terminus phosphorylated peptide of m/z 753.997 showing 3 distinct elution peaks for 3 distinct phosphorylation sites. ( D ) Representative MS/MS spectrum of the phosphorylated C-terminus peptide at position 368. The confidence of modification sites is estimated by an Ascore (Ascore = 20 for P value of .01).

    Techniques Used: Recombinant, In Vitro, Autoradiography, Tandem Mass Spectroscopy, Software, Modification, Labeling

    Inhibition of CK2 enhances ROS production by NOX1 in colon T84 epithelial cells under inflammatory conditions. ( A ) ROS production was measured by chemiluminescence in T84 cells co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 after 24-hour incubation at 37°C. n = 3. ( B ) CK2 activity (top) assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody and NOXO1 expression (middle) in T84 cells co-stimulated as in (A) . Representative of 3 independent experiments. ( C ) Concentration-dependent effect of CX-4549 on ROS production by NOX1 in T84 cells co-stimulated as in (A) in presence or absence of various concentrations of CX-4945 for 24 hours at 37°C. Data were expressed as percentage of control (cells treated with TNFα + IL17 in absence of CX-4549); n = 7 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗ P < .01. ( D ) Concentration-dependent effect of TBBz on ROS production by NOX1 measured by chemiluminescence in T84 cells co-stimulated as in (A) in the presence or absence of various concentrations of TBBz for 24 hours at 37°C. ( E ) Concentration-dependent effect of CX4945 on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM; one-way ANOVA with Dunnett multiple comparisons test; ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. ( F ) Concentration-dependent effect of TBBz on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM.
    Figure Legend Snippet: Inhibition of CK2 enhances ROS production by NOX1 in colon T84 epithelial cells under inflammatory conditions. ( A ) ROS production was measured by chemiluminescence in T84 cells co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 after 24-hour incubation at 37°C. n = 3. ( B ) CK2 activity (top) assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody and NOXO1 expression (middle) in T84 cells co-stimulated as in (A) . Representative of 3 independent experiments. ( C ) Concentration-dependent effect of CX-4549 on ROS production by NOX1 in T84 cells co-stimulated as in (A) in presence or absence of various concentrations of CX-4945 for 24 hours at 37°C. Data were expressed as percentage of control (cells treated with TNFα + IL17 in absence of CX-4549); n = 7 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗ P < .01. ( D ) Concentration-dependent effect of TBBz on ROS production by NOX1 measured by chemiluminescence in T84 cells co-stimulated as in (A) in the presence or absence of various concentrations of TBBz for 24 hours at 37°C. ( E ) Concentration-dependent effect of CX4945 on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM; one-way ANOVA with Dunnett multiple comparisons test; ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. ( F ) Concentration-dependent effect of TBBz on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM.

    Techniques Used: Inhibition, Incubation, Activity Assay, Expressing, Concentration Assay

    Inhibition of CK2 enhances ROS production by NOX1 in colon organoids under inflammatory conditions. ( A ) Representative immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon organoids stimulated as in <xref ref-type=Figure 1 A for 24 hours at 37°C. ( B ) Representative images of colon organoids established from colon biopsies of control patients. At day 8, organoids were co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 for 24 hours at 37°C. Scale bar: 100 μm. ( C ) ROS production was measured by chemiluminescence in colon organoids treated as in (B). ( D ) Data as in (C) were quantified and expressed as percentage of control (resting cells in the absence of CX-4549); n = 4 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05, ∗∗∗ P < .001. ( E ) ROS production by organoids treated as in ( B) was assessed by NBT reduction. Phase contrast microcopy with ×400 magnification. " title="Inhibition of CK2 enhances ROS production by NOX1 in colon organoids ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Inhibition of CK2 enhances ROS production by NOX1 in colon organoids under inflammatory conditions. ( A ) Representative immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon organoids stimulated as in Figure 1 A for 24 hours at 37°C. ( B ) Representative images of colon organoids established from colon biopsies of control patients. At day 8, organoids were co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 for 24 hours at 37°C. Scale bar: 100 μm. ( C ) ROS production was measured by chemiluminescence in colon organoids treated as in (B). ( D ) Data as in (C) were quantified and expressed as percentage of control (resting cells in the absence of CX-4549); n = 4 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05, ∗∗∗ P < .001. ( E ) ROS production by organoids treated as in ( B) was assessed by NBT reduction. Phase contrast microcopy with ×400 magnification.

    Techniques Used: Inhibition, Western Blot

    CK2 activity is highly reduced and NOX1 expression is increased during TNBS-induced acute colitis. ( A ) Left, representative image of colons 24 hours after injection of TNBS or vehicle (CTL); middle, weight/length ratio changes after TNBS treatment; right, macroscopic lesions as assessed by Wallace score. Mean ± SEM, ≥10 animals; Mann-Whitney test; ∗∗∗∗ P < .0001. ( B ) Histologic images of colons 24 hours after TNBS-induced colitis (original magnification, ×200) as compared with CTL (vehicle). Scale bar: 50 μm. ( C ) CK2 activity in cytosolic and membrane fractions of colon homogenates assessed 24 hours after injection of TNBS or vehicle (CTL) with the phospho-CK2-substrate antibody. Four mice are shown (M1 to M4) for each group. ( D ) Densitometry analysis of p-CK2 substrates normalized to β-actin expression. Mean ± SEM from 10 animals; Kruskal-Wallis test with Dunn multiple comparisons test; ∗∗ P < .01, ∗∗∗ P < .001. ( E ) Immunoblots of CK2α/α′ and CK2β in colon homogenates 24 hours after injection of TNBS or vehicle (CTL). Four mice are shown (M1 to M4) for each group. ( F ) Densitometry analysis of CK2α/α′ and CK2β normalized to β-actin expression. Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗∗ P < .01. ( G ) Immunoblots of NOX1 and p22 PHOX in colon homogenates 24 hours after injection of TNBS or vehicle (CTL). Two mice (M1 to M2) in the CTL group and 5 mice (M1 to M5) in the TNBS group are shown. ( H ) Densitometry analysis of NOX1 and p22 PHOX normalized to β-actin expression. Mean ± SEM, ≥8 animals; Mann-Whitney test; ∗∗ P < .01. ( I ) Expression of NOXO1 and NOXA1 RNA messengers during TNBS-induced acute colitis. Relative expression levels for each gene were calculated using the 2 -ΔΔCt method, with normalization to the average and GAPDH housekeeping genes. Mean ± SEM, 9 animals; Mann-Whitney test; ∗∗ P < .01.
    Figure Legend Snippet: CK2 activity is highly reduced and NOX1 expression is increased during TNBS-induced acute colitis. ( A ) Left, representative image of colons 24 hours after injection of TNBS or vehicle (CTL); middle, weight/length ratio changes after TNBS treatment; right, macroscopic lesions as assessed by Wallace score. Mean ± SEM, ≥10 animals; Mann-Whitney test; ∗∗∗∗ P < .0001. ( B ) Histologic images of colons 24 hours after TNBS-induced colitis (original magnification, ×200) as compared with CTL (vehicle). Scale bar: 50 μm. ( C ) CK2 activity in cytosolic and membrane fractions of colon homogenates assessed 24 hours after injection of TNBS or vehicle (CTL) with the phospho-CK2-substrate antibody. Four mice are shown (M1 to M4) for each group. ( D ) Densitometry analysis of p-CK2 substrates normalized to β-actin expression. Mean ± SEM from 10 animals; Kruskal-Wallis test with Dunn multiple comparisons test; ∗∗ P < .01, ∗∗∗ P < .001. ( E ) Immunoblots of CK2α/α′ and CK2β in colon homogenates 24 hours after injection of TNBS or vehicle (CTL). Four mice are shown (M1 to M4) for each group. ( F ) Densitometry analysis of CK2α/α′ and CK2β normalized to β-actin expression. Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗∗ P < .01. ( G ) Immunoblots of NOX1 and p22 PHOX in colon homogenates 24 hours after injection of TNBS or vehicle (CTL). Two mice (M1 to M2) in the CTL group and 5 mice (M1 to M5) in the TNBS group are shown. ( H ) Densitometry analysis of NOX1 and p22 PHOX normalized to β-actin expression. Mean ± SEM, ≥8 animals; Mann-Whitney test; ∗∗ P < .01. ( I ) Expression of NOXO1 and NOXA1 RNA messengers during TNBS-induced acute colitis. Relative expression levels for each gene were calculated using the 2 -ΔΔCt method, with normalization to the average and GAPDH housekeeping genes. Mean ± SEM, 9 animals; Mann-Whitney test; ∗∗ P < .01.

    Techniques Used: Activity Assay, Expressing, Injection, MANN-WHITNEY, Western Blot

    Effect of CK2β and CK2α overexpression on CK2 activity and ROS production in colon T84 epithelial cells. ( A ) Immunoblots of HA-CK2β and Myc-CK2α overexpressed individually in colon T84 cells. ( B ) CK2 activity assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody in T84 cells overexpressing CK2β or CK2α. ( C ) ROS production was measured by chemiluminescence in T84 cells overexpressing CK2β or CK2α. n = 3. ( D ) Effect of increasing concentrations of recombinant CK2β subunit on phosphorylation of NOXO1 by CK2 in vitro . Autoradiography (Autorad.) and Ponceau-Red are shown.
    Figure Legend Snippet: Effect of CK2β and CK2α overexpression on CK2 activity and ROS production in colon T84 epithelial cells. ( A ) Immunoblots of HA-CK2β and Myc-CK2α overexpressed individually in colon T84 cells. ( B ) CK2 activity assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody in T84 cells overexpressing CK2β or CK2α. ( C ) ROS production was measured by chemiluminescence in T84 cells overexpressing CK2β or CK2α. n = 3. ( D ) Effect of increasing concentrations of recombinant CK2β subunit on phosphorylation of NOXO1 by CK2 in vitro . Autoradiography (Autorad.) and Ponceau-Red are shown.

    Techniques Used: Over Expression, Activity Assay, Western Blot, Recombinant, In Vitro, Autoradiography

    The highly selective CK2 inhibitor CX-4945 exacerbates TNBS-induced colitis. ( A ) Representative images of colons 24 hours after injection of TNBS or vehicle (CTL) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. ( B ) Histologic images of colons 24 hours after TNBS-induced colitis (original magnification, ×200) as compared with CTL (vehicle) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. Scale bar: 50 μm. ( C ) Histologic damage of colon as assessed by the Ameho score in mice treated as in (B) . Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗ P < .05. ( D ) Secretion of CXCL1 (pg. mg -1 protein) in colons of mice treated as in (B). Mean ± SEM from 4 animals (CTL groups with and without CX-4945) to 6 animals (TNBS groups with and without CX-4945), Mann-Whitney test; ∗ P < .05. ( E ) ROS production assessed ex vivo by NBT reduction in colon tissue of mice treated as in (B) . Formazan deposits were examined macroscopically. Representative of 4 mice in the CTL groups with and without CX-4945 and at least 7 mice in the TNBS groups with and without CX-4945. ( F ) MDA (nmol. mg -1 protein) content in the colon of mice treated as in (B) , normalized as compared with control. Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗ P < .05.
    Figure Legend Snippet: The highly selective CK2 inhibitor CX-4945 exacerbates TNBS-induced colitis. ( A ) Representative images of colons 24 hours after injection of TNBS or vehicle (CTL) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. ( B ) Histologic images of colons 24 hours after TNBS-induced colitis (original magnification, ×200) as compared with CTL (vehicle) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. Scale bar: 50 μm. ( C ) Histologic damage of colon as assessed by the Ameho score in mice treated as in (B) . Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗ P < .05. ( D ) Secretion of CXCL1 (pg. mg -1 protein) in colons of mice treated as in (B). Mean ± SEM from 4 animals (CTL groups with and without CX-4945) to 6 animals (TNBS groups with and without CX-4945), Mann-Whitney test; ∗ P < .05. ( E ) ROS production assessed ex vivo by NBT reduction in colon tissue of mice treated as in (B) . Formazan deposits were examined macroscopically. Representative of 4 mice in the CTL groups with and without CX-4945 and at least 7 mice in the TNBS groups with and without CX-4945. ( F ) MDA (nmol. mg -1 protein) content in the colon of mice treated as in (B) , normalized as compared with control. Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗ P < .05.

    Techniques Used: Injection, MANN-WHITNEY, Ex Vivo

    Expression of NOX1 and p22 PHOX during TNBS-induced acute colitis in the presence or absence of CX-4945. Immunoblots of NOX1 and p22 PHOX in colon homogenates 24 hours after injection of TNBS or vehicle (CTL) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. Results from 2 mice (M1 to M2) in the CTL group and 4 mice (M1 to M4) in the TNBS group are shown.
    Figure Legend Snippet: Expression of NOX1 and p22 PHOX during TNBS-induced acute colitis in the presence or absence of CX-4945. Immunoblots of NOX1 and p22 PHOX in colon homogenates 24 hours after injection of TNBS or vehicle (CTL) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. Results from 2 mice (M1 to M2) in the CTL group and 4 mice (M1 to M4) in the TNBS group are shown.

    Techniques Used: Expressing, Western Blot, Injection

    phospho ck2 substrate  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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  • 94

    Structured Review

    Cell Signaling Technology Inc phospho ck2 substrate
    Proteins Co-Immunoprecipitating With NOXO1 in T84 Cells Stimulated by TNFα (5 ng/mL) + IL17 (50 ng/mL) and Related to NADPH Oxidase or Redox Signaling
    Phospho Ck2 Substrate, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho ck2 substrate/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho ck2 substrate - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation"

    Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2022.01.003

    Proteins Co-Immunoprecipitating With NOXO1 in T84 Cells Stimulated by TNFα (5 ng/mL) + IL17 (50 ng/mL) and Related to NADPH Oxidase or Redox Signaling
    Figure Legend Snippet: Proteins Co-Immunoprecipitating With NOXO1 in T84 Cells Stimulated by TNFα (5 ng/mL) + IL17 (50 ng/mL) and Related to NADPH Oxidase or Redox Signaling

    Techniques Used: Binding Assay, Protein Binding

    CK2 interacts with NOXO1 in T84 colon epithelial cells under inflammatory conditions. ( A ) Immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon T84 cells stimulated with TNFα (5 ng/mL) or IL17 (50 ng/mL) individually or in combination for 24 hours at 37°C. Representative of 3 independent experiments. ( B ) ROS production was measured by chemiluminescence in T84 cells stimulated as in (A) . n = 3. ( C ) Immunoblots of CK2 α/α′ in NOXO1 IPP from T84 cells stimulated as in (A) . Representative of 4 independent experiments. ( D ) Densitometry analysis of CK2 α/α′ in NOXO1 IPP as in ( C ), normalized to CK2 α/α′expression in cell lysates; n = 4; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05. ( E ) Confocal microcopy of T84 cells co-stimulated or not with TNFα + IL17 for 24 hours at 37°C. NOXO1 ( green ), CK2 α/α′ ( red ), DAPI ( blue ). Scale bar: 10 μm. Representative of 6 independent experiments.
    Figure Legend Snippet: CK2 interacts with NOXO1 in T84 colon epithelial cells under inflammatory conditions. ( A ) Immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon T84 cells stimulated with TNFα (5 ng/mL) or IL17 (50 ng/mL) individually or in combination for 24 hours at 37°C. Representative of 3 independent experiments. ( B ) ROS production was measured by chemiluminescence in T84 cells stimulated as in (A) . n = 3. ( C ) Immunoblots of CK2 α/α′ in NOXO1 IPP from T84 cells stimulated as in (A) . Representative of 4 independent experiments. ( D ) Densitometry analysis of CK2 α/α′ in NOXO1 IPP as in ( C ), normalized to CK2 α/α′expression in cell lysates; n = 4; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05. ( E ) Confocal microcopy of T84 cells co-stimulated or not with TNFα + IL17 for 24 hours at 37°C. NOXO1 ( green ), CK2 α/α′ ( red ), DAPI ( blue ). Scale bar: 10 μm. Representative of 6 independent experiments.

    Techniques Used: Western Blot, Expressing

    CK2 interacts directly with NOXO1 through the N-terminal region mostly containing the PX domain. ( A ) Coomassie Blue staining of recombinant NOXO1, p47 PHOX , NOXA1, and p67 PHOX . ( B ) Dot-blot analysis of interaction between CK2 and recombinant NOXO1, p47 PHOX , NOXA1, or p67 PHOX . Representative of 3 independent experiments. ( C ) Schematic representation and Coomassie Blue staining of GST fusion proteins of the NOXO1 full-length (β isoform), N-terminal (1-157), and C-terminal regions (232-371). ( D ) Pull-down of CK2 from resting T84 cells lysates by full-length GST-NOXO1, GST-NOXO1 (1-157), GST-NOXO1 (232-371), or GST (control). Representative of 3 independent experiments. ( E ) Densitometry analysis of CK2 α/α′ pull-downed as in ( D ), normalized to GST-fusion proteins; n = 3; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗∗∗ P < 0.0001.
    Figure Legend Snippet: CK2 interacts directly with NOXO1 through the N-terminal region mostly containing the PX domain. ( A ) Coomassie Blue staining of recombinant NOXO1, p47 PHOX , NOXA1, and p67 PHOX . ( B ) Dot-blot analysis of interaction between CK2 and recombinant NOXO1, p47 PHOX , NOXA1, or p67 PHOX . Representative of 3 independent experiments. ( C ) Schematic representation and Coomassie Blue staining of GST fusion proteins of the NOXO1 full-length (β isoform), N-terminal (1-157), and C-terminal regions (232-371). ( D ) Pull-down of CK2 from resting T84 cells lysates by full-length GST-NOXO1, GST-NOXO1 (1-157), GST-NOXO1 (232-371), or GST (control). Representative of 3 independent experiments. ( E ) Densitometry analysis of CK2 α/α′ pull-downed as in ( D ), normalized to GST-fusion proteins; n = 3; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗∗∗ P < 0.0001.

    Techniques Used: Staining, Recombinant, Dot Blot

    CK2 phosphorylates recombinant NOXO1 in vitro on several sites. ( A ) Autoradiography of recombinant NOXO1 phosphorylated with constitutively active CK2 in the presence of radioactive [γ -32P ] ATP. Time course ( left ), with or without CK2 ( right ). Representative of 3 independent experiments. ( B ) MS/MS protein coverage of recombinant NOXO1 after in vitro phosphorylation by CK2. Peptides were filtered according to 1% false discovery rate that corresponds to an identification score value >37.6 (–10lgP) with the software Peaks Studio Xpro. The confidence of modification sites is estimated by an Ascore, which calculates an ambiguity score as –10 × log 10 (p). The P value indicates the likelihood that the peptide is matched by chance (Ascore = 20 for P value of .01). Only confident modification sites with Ascore >20 were retained and their position labeled ( red ). ( C ) XIC of the heavily C-terminus phosphorylated peptide of m/z 753.997 showing 3 distinct elution peaks for 3 distinct phosphorylation sites. ( D ) Representative MS/MS spectrum of the phosphorylated C-terminus peptide at position 368. The confidence of modification sites is estimated by an Ascore (Ascore = 20 for P value of .01).
    Figure Legend Snippet: CK2 phosphorylates recombinant NOXO1 in vitro on several sites. ( A ) Autoradiography of recombinant NOXO1 phosphorylated with constitutively active CK2 in the presence of radioactive [γ -32P ] ATP. Time course ( left ), with or without CK2 ( right ). Representative of 3 independent experiments. ( B ) MS/MS protein coverage of recombinant NOXO1 after in vitro phosphorylation by CK2. Peptides were filtered according to 1% false discovery rate that corresponds to an identification score value >37.6 (–10lgP) with the software Peaks Studio Xpro. The confidence of modification sites is estimated by an Ascore, which calculates an ambiguity score as –10 × log 10 (p). The P value indicates the likelihood that the peptide is matched by chance (Ascore = 20 for P value of .01). Only confident modification sites with Ascore >20 were retained and their position labeled ( red ). ( C ) XIC of the heavily C-terminus phosphorylated peptide of m/z 753.997 showing 3 distinct elution peaks for 3 distinct phosphorylation sites. ( D ) Representative MS/MS spectrum of the phosphorylated C-terminus peptide at position 368. The confidence of modification sites is estimated by an Ascore (Ascore = 20 for P value of .01).

    Techniques Used: Recombinant, In Vitro, Autoradiography, Tandem Mass Spectroscopy, Software, Modification, Labeling

    Inhibition of CK2 enhances ROS production by NOX1 in colon T84 epithelial cells under inflammatory conditions. ( A ) ROS production was measured by chemiluminescence in T84 cells co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 after 24-hour incubation at 37°C. n = 3. ( B ) CK2 activity (top) assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody and NOXO1 expression (middle) in T84 cells co-stimulated as in (A) . Representative of 3 independent experiments. ( C ) Concentration-dependent effect of CX-4549 on ROS production by NOX1 in T84 cells co-stimulated as in (A) in presence or absence of various concentrations of CX-4945 for 24 hours at 37°C. Data were expressed as percentage of control (cells treated with TNFα + IL17 in absence of CX-4549); n = 7 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗ P < .01. ( D ) Concentration-dependent effect of TBBz on ROS production by NOX1 measured by chemiluminescence in T84 cells co-stimulated as in (A) in the presence or absence of various concentrations of TBBz for 24 hours at 37°C. ( E ) Concentration-dependent effect of CX4945 on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM; one-way ANOVA with Dunnett multiple comparisons test; ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. ( F ) Concentration-dependent effect of TBBz on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM.
    Figure Legend Snippet: Inhibition of CK2 enhances ROS production by NOX1 in colon T84 epithelial cells under inflammatory conditions. ( A ) ROS production was measured by chemiluminescence in T84 cells co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 after 24-hour incubation at 37°C. n = 3. ( B ) CK2 activity (top) assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody and NOXO1 expression (middle) in T84 cells co-stimulated as in (A) . Representative of 3 independent experiments. ( C ) Concentration-dependent effect of CX-4549 on ROS production by NOX1 in T84 cells co-stimulated as in (A) in presence or absence of various concentrations of CX-4945 for 24 hours at 37°C. Data were expressed as percentage of control (cells treated with TNFα + IL17 in absence of CX-4549); n = 7 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗ P < .01. ( D ) Concentration-dependent effect of TBBz on ROS production by NOX1 measured by chemiluminescence in T84 cells co-stimulated as in (A) in the presence or absence of various concentrations of TBBz for 24 hours at 37°C. ( E ) Concentration-dependent effect of CX4945 on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM; one-way ANOVA with Dunnett multiple comparisons test; ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. ( F ) Concentration-dependent effect of TBBz on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM.

    Techniques Used: Inhibition, Incubation, Activity Assay, Expressing, Concentration Assay

    Inhibition of CK2 enhances ROS production by NOX1 in colon organoids under inflammatory conditions. ( A ) Representative immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon organoids stimulated as in <xref ref-type=Figure 1 A for 24 hours at 37°C. ( B ) Representative images of colon organoids established from colon biopsies of control patients. At day 8, organoids were co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 for 24 hours at 37°C. Scale bar: 100 μm. ( C ) ROS production was measured by chemiluminescence in colon organoids treated as in (B). ( D ) Data as in (C) were quantified and expressed as percentage of control (resting cells in the absence of CX-4549); n = 4 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05, ∗∗∗ P < .001. ( E ) ROS production by organoids treated as in ( B) was assessed by NBT reduction. Phase contrast microcopy with ×400 magnification. " title="Inhibition of CK2 enhances ROS production by NOX1 in colon organoids ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Inhibition of CK2 enhances ROS production by NOX1 in colon organoids under inflammatory conditions. ( A ) Representative immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon organoids stimulated as in Figure 1 A for 24 hours at 37°C. ( B ) Representative images of colon organoids established from colon biopsies of control patients. At day 8, organoids were co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 for 24 hours at 37°C. Scale bar: 100 μm. ( C ) ROS production was measured by chemiluminescence in colon organoids treated as in (B). ( D ) Data as in (C) were quantified and expressed as percentage of control (resting cells in the absence of CX-4549); n = 4 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05, ∗∗∗ P < .001. ( E ) ROS production by organoids treated as in ( B) was assessed by NBT reduction. Phase contrast microcopy with ×400 magnification.

    Techniques Used: Inhibition, Western Blot

    CK2 activity is highly reduced and NOX1 expression is increased during TNBS-induced acute colitis. ( A ) Left, representative image of colons 24 hours after injection of TNBS or vehicle (CTL); middle, weight/length ratio changes after TNBS treatment; right, macroscopic lesions as assessed by Wallace score. Mean ± SEM, ≥10 animals; Mann-Whitney test; ∗∗∗∗ P < .0001. ( B ) Histologic images of colons 24 hours after TNBS-induced colitis (original magnification, ×200) as compared with CTL (vehicle). Scale bar: 50 μm. ( C ) CK2 activity in cytosolic and membrane fractions of colon homogenates assessed 24 hours after injection of TNBS or vehicle (CTL) with the phospho-CK2-substrate antibody. Four mice are shown (M1 to M4) for each group. ( D ) Densitometry analysis of p-CK2 substrates normalized to β-actin expression. Mean ± SEM from 10 animals; Kruskal-Wallis test with Dunn multiple comparisons test; ∗∗ P < .01, ∗∗∗ P < .001. ( E ) Immunoblots of CK2α/α′ and CK2β in colon homogenates 24 hours after injection of TNBS or vehicle (CTL). Four mice are shown (M1 to M4) for each group. ( F ) Densitometry analysis of CK2α/α′ and CK2β normalized to β-actin expression. Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗∗ P < .01. ( G ) Immunoblots of NOX1 and p22 PHOX in colon homogenates 24 hours after injection of TNBS or vehicle (CTL). Two mice (M1 to M2) in the CTL group and 5 mice (M1 to M5) in the TNBS group are shown. ( H ) Densitometry analysis of NOX1 and p22 PHOX normalized to β-actin expression. Mean ± SEM, ≥8 animals; Mann-Whitney test; ∗∗ P < .01. ( I ) Expression of NOXO1 and NOXA1 RNA messengers during TNBS-induced acute colitis. Relative expression levels for each gene were calculated using the 2 -ΔΔCt method, with normalization to the average and GAPDH housekeeping genes. Mean ± SEM, 9 animals; Mann-Whitney test; ∗∗ P < .01.
    Figure Legend Snippet: CK2 activity is highly reduced and NOX1 expression is increased during TNBS-induced acute colitis. ( A ) Left, representative image of colons 24 hours after injection of TNBS or vehicle (CTL); middle, weight/length ratio changes after TNBS treatment; right, macroscopic lesions as assessed by Wallace score. Mean ± SEM, ≥10 animals; Mann-Whitney test; ∗∗∗∗ P < .0001. ( B ) Histologic images of colons 24 hours after TNBS-induced colitis (original magnification, ×200) as compared with CTL (vehicle). Scale bar: 50 μm. ( C ) CK2 activity in cytosolic and membrane fractions of colon homogenates assessed 24 hours after injection of TNBS or vehicle (CTL) with the phospho-CK2-substrate antibody. Four mice are shown (M1 to M4) for each group. ( D ) Densitometry analysis of p-CK2 substrates normalized to β-actin expression. Mean ± SEM from 10 animals; Kruskal-Wallis test with Dunn multiple comparisons test; ∗∗ P < .01, ∗∗∗ P < .001. ( E ) Immunoblots of CK2α/α′ and CK2β in colon homogenates 24 hours after injection of TNBS or vehicle (CTL). Four mice are shown (M1 to M4) for each group. ( F ) Densitometry analysis of CK2α/α′ and CK2β normalized to β-actin expression. Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗∗ P < .01. ( G ) Immunoblots of NOX1 and p22 PHOX in colon homogenates 24 hours after injection of TNBS or vehicle (CTL). Two mice (M1 to M2) in the CTL group and 5 mice (M1 to M5) in the TNBS group are shown. ( H ) Densitometry analysis of NOX1 and p22 PHOX normalized to β-actin expression. Mean ± SEM, ≥8 animals; Mann-Whitney test; ∗∗ P < .01. ( I ) Expression of NOXO1 and NOXA1 RNA messengers during TNBS-induced acute colitis. Relative expression levels for each gene were calculated using the 2 -ΔΔCt method, with normalization to the average and GAPDH housekeeping genes. Mean ± SEM, 9 animals; Mann-Whitney test; ∗∗ P < .01.

    Techniques Used: Activity Assay, Expressing, Injection, MANN-WHITNEY, Western Blot

    Effect of CK2β and CK2α overexpression on CK2 activity and ROS production in colon T84 epithelial cells. ( A ) Immunoblots of HA-CK2β and Myc-CK2α overexpressed individually in colon T84 cells. ( B ) CK2 activity assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody in T84 cells overexpressing CK2β or CK2α. ( C ) ROS production was measured by chemiluminescence in T84 cells overexpressing CK2β or CK2α. n = 3. ( D ) Effect of increasing concentrations of recombinant CK2β subunit on phosphorylation of NOXO1 by CK2 in vitro . Autoradiography (Autorad.) and Ponceau-Red are shown.
    Figure Legend Snippet: Effect of CK2β and CK2α overexpression on CK2 activity and ROS production in colon T84 epithelial cells. ( A ) Immunoblots of HA-CK2β and Myc-CK2α overexpressed individually in colon T84 cells. ( B ) CK2 activity assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody in T84 cells overexpressing CK2β or CK2α. ( C ) ROS production was measured by chemiluminescence in T84 cells overexpressing CK2β or CK2α. n = 3. ( D ) Effect of increasing concentrations of recombinant CK2β subunit on phosphorylation of NOXO1 by CK2 in vitro . Autoradiography (Autorad.) and Ponceau-Red are shown.

    Techniques Used: Over Expression, Activity Assay, Western Blot, Recombinant, In Vitro, Autoradiography

    The highly selective CK2 inhibitor CX-4945 exacerbates TNBS-induced colitis. ( A ) Representative images of colons 24 hours after injection of TNBS or vehicle (CTL) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. ( B ) Histologic images of colons 24 hours after TNBS-induced colitis (original magnification, ×200) as compared with CTL (vehicle) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. Scale bar: 50 μm. ( C ) Histologic damage of colon as assessed by the Ameho score in mice treated as in (B) . Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗ P < .05. ( D ) Secretion of CXCL1 (pg. mg -1 protein) in colons of mice treated as in (B). Mean ± SEM from 4 animals (CTL groups with and without CX-4945) to 6 animals (TNBS groups with and without CX-4945), Mann-Whitney test; ∗ P < .05. ( E ) ROS production assessed ex vivo by NBT reduction in colon tissue of mice treated as in (B) . Formazan deposits were examined macroscopically. Representative of 4 mice in the CTL groups with and without CX-4945 and at least 7 mice in the TNBS groups with and without CX-4945. ( F ) MDA (nmol. mg -1 protein) content in the colon of mice treated as in (B) , normalized as compared with control. Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗ P < .05.
    Figure Legend Snippet: The highly selective CK2 inhibitor CX-4945 exacerbates TNBS-induced colitis. ( A ) Representative images of colons 24 hours after injection of TNBS or vehicle (CTL) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. ( B ) Histologic images of colons 24 hours after TNBS-induced colitis (original magnification, ×200) as compared with CTL (vehicle) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. Scale bar: 50 μm. ( C ) Histologic damage of colon as assessed by the Ameho score in mice treated as in (B) . Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗ P < .05. ( D ) Secretion of CXCL1 (pg. mg -1 protein) in colons of mice treated as in (B). Mean ± SEM from 4 animals (CTL groups with and without CX-4945) to 6 animals (TNBS groups with and without CX-4945), Mann-Whitney test; ∗ P < .05. ( E ) ROS production assessed ex vivo by NBT reduction in colon tissue of mice treated as in (B) . Formazan deposits were examined macroscopically. Representative of 4 mice in the CTL groups with and without CX-4945 and at least 7 mice in the TNBS groups with and without CX-4945. ( F ) MDA (nmol. mg -1 protein) content in the colon of mice treated as in (B) , normalized as compared with control. Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗ P < .05.

    Techniques Used: Injection, MANN-WHITNEY, Ex Vivo

    Expression of NOX1 and p22 PHOX during TNBS-induced acute colitis in the presence or absence of CX-4945. Immunoblots of NOX1 and p22 PHOX in colon homogenates 24 hours after injection of TNBS or vehicle (CTL) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. Results from 2 mice (M1 to M2) in the CTL group and 4 mice (M1 to M4) in the TNBS group are shown.
    Figure Legend Snippet: Expression of NOX1 and p22 PHOX during TNBS-induced acute colitis in the presence or absence of CX-4945. Immunoblots of NOX1 and p22 PHOX in colon homogenates 24 hours after injection of TNBS or vehicle (CTL) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. Results from 2 mice (M1 to M2) in the CTL group and 4 mice (M1 to M4) in the TNBS group are shown.

    Techniques Used: Expressing, Western Blot, Injection

    phospho ser thr ck2 substrate rabbit  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho ser thr ck2 substrate rabbit
    List of commercial primary antibodies used for Western blot.
    Phospho Ser Thr Ck2 Substrate Rabbit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "GPR68 Contributes to Persistent Acidosis-Induced Activation of AGC Kinases and Tyrosine Phosphorylation in Organotypic Hippocampal Slices"

    Article Title: GPR68 Contributes to Persistent Acidosis-Induced Activation of AGC Kinases and Tyrosine Phosphorylation in Organotypic Hippocampal Slices

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2021.692217

    List of commercial primary antibodies used for Western blot.
    Figure Legend Snippet: List of commercial primary antibodies used for Western blot.

    Techniques Used: Western Blot

    s t  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc s t
    List of commercial primary antibodies used for Western blot.
    S T, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "GPR68 Contributes to Persistent Acidosis-Induced Activation of AGC Kinases and Tyrosine Phosphorylation in Organotypic Hippocampal Slices"

    Article Title: GPR68 Contributes to Persistent Acidosis-Induced Activation of AGC Kinases and Tyrosine Phosphorylation in Organotypic Hippocampal Slices

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2021.692217

    List of commercial primary antibodies used for Western blot.
    Figure Legend Snippet: List of commercial primary antibodies used for Western blot.

    Techniques Used: Western Blot

    ck2 immunoreaction  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ck2 immunoreaction
    <t>CK2</t> inhibition exerts disease modifying properties. (A) Time frame of the experiment. TBB was administered four times in vivo on days “−3” (i.e., 3 days before status epilepticus) through “0” (i.e., day of pilocarpine-induced status epilepticus). All experiments thereafter were performed during the chronic stage of recurrent seizures. (B–D) Latency to the first stage 4 or 5 seizure after pilocarpine injection in vehicle-pretreated (B) and TBB-pretreated (C) rats. The median latency was significantly (*** P < 0.001, Mann-Whitney U-test) prolonged in the TBB-pretreated group (D) . (E) Number of interictal spikes per hour (left y -axis) in the electroencephalogram (EEG) during 3 days (97–100 post-SE). Note that the number of daily seizures during this period is also depicted (diamonds, right y -axis). There was a significant group effect of TBB pretreatment (** P < 0.01, two-way ANOVA with Tukey post hoc test). (F) Number of generalized seizures (at least stage 3) at daylight (from 06:00 to 18:00 h) for both animal groups as grand average for the entire recording period. (G) Seizure burden progression as fold change of seizure rates during epileptogenesis. The variability was significantly reduced in TBB-pretreated epileptic animals (** P < 0.01, F -test). (H) Relative risk reduction (RRR) of seizure progression by TBB pretreatment was significant (* P < 0.05, t -test).
    Ck2 Immunoreaction, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "CK2 Inhibition Prior to Status Epilepticus Persistently Enhances K Ca 2 Function in CA1 Which Slows Down Disease Progression"

    Article Title: CK2 Inhibition Prior to Status Epilepticus Persistently Enhances K Ca 2 Function in CA1 Which Slows Down Disease Progression

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2020.00033

    CK2 inhibition exerts disease modifying properties. (A) Time frame of the experiment. TBB was administered four times in vivo on days “−3” (i.e., 3 days before status epilepticus) through “0” (i.e., day of pilocarpine-induced status epilepticus). All experiments thereafter were performed during the chronic stage of recurrent seizures. (B–D) Latency to the first stage 4 or 5 seizure after pilocarpine injection in vehicle-pretreated (B) and TBB-pretreated (C) rats. The median latency was significantly (*** P < 0.001, Mann-Whitney U-test) prolonged in the TBB-pretreated group (D) . (E) Number of interictal spikes per hour (left y -axis) in the electroencephalogram (EEG) during 3 days (97–100 post-SE). Note that the number of daily seizures during this period is also depicted (diamonds, right y -axis). There was a significant group effect of TBB pretreatment (** P < 0.01, two-way ANOVA with Tukey post hoc test). (F) Number of generalized seizures (at least stage 3) at daylight (from 06:00 to 18:00 h) for both animal groups as grand average for the entire recording period. (G) Seizure burden progression as fold change of seizure rates during epileptogenesis. The variability was significantly reduced in TBB-pretreated epileptic animals (** P < 0.01, F -test). (H) Relative risk reduction (RRR) of seizure progression by TBB pretreatment was significant (* P < 0.05, t -test).
    Figure Legend Snippet: CK2 inhibition exerts disease modifying properties. (A) Time frame of the experiment. TBB was administered four times in vivo on days “−3” (i.e., 3 days before status epilepticus) through “0” (i.e., day of pilocarpine-induced status epilepticus). All experiments thereafter were performed during the chronic stage of recurrent seizures. (B–D) Latency to the first stage 4 or 5 seizure after pilocarpine injection in vehicle-pretreated (B) and TBB-pretreated (C) rats. The median latency was significantly (*** P < 0.001, Mann-Whitney U-test) prolonged in the TBB-pretreated group (D) . (E) Number of interictal spikes per hour (left y -axis) in the electroencephalogram (EEG) during 3 days (97–100 post-SE). Note that the number of daily seizures during this period is also depicted (diamonds, right y -axis). There was a significant group effect of TBB pretreatment (** P < 0.01, two-way ANOVA with Tukey post hoc test). (F) Number of generalized seizures (at least stage 3) at daylight (from 06:00 to 18:00 h) for both animal groups as grand average for the entire recording period. (G) Seizure burden progression as fold change of seizure rates during epileptogenesis. The variability was significantly reduced in TBB-pretreated epileptic animals (** P < 0.01, F -test). (H) Relative risk reduction (RRR) of seizure progression by TBB pretreatment was significant (* P < 0.05, t -test).

    Techniques Used: Inhibition, In Vivo, Injection, MANN-WHITNEY

    CK2 inhibition facilitates CA1 plasticity. (A) Sample traces of CA1 stratum radiatum field excitatory postsynaptic potentials (fEPSP) following Schaffer collateral stimulation in control (A 1 ) and epileptic animals (A 2 ). (B) Input–output curves of all four groups without significant group differences. (C) Paired-pulse ratios (PPRs) were similar among all four experimental groups. Short-term plasticity during five stimuli at frequencies of 10, 25, and 33 Hz (D) , and subsequent changes of fEPSP slope (E) and PPR (F) after this short-term plasticity paradigm. Only slices from the TBB-pretreated epileptic group showed significantly facilitated plasticity. * P < 0.05.
    Figure Legend Snippet: CK2 inhibition facilitates CA1 plasticity. (A) Sample traces of CA1 stratum radiatum field excitatory postsynaptic potentials (fEPSP) following Schaffer collateral stimulation in control (A 1 ) and epileptic animals (A 2 ). (B) Input–output curves of all four groups without significant group differences. (C) Paired-pulse ratios (PPRs) were similar among all four experimental groups. Short-term plasticity during five stimuli at frequencies of 10, 25, and 33 Hz (D) , and subsequent changes of fEPSP slope (E) and PPR (F) after this short-term plasticity paradigm. Only slices from the TBB-pretreated epileptic group showed significantly facilitated plasticity. * P < 0.05.

    Techniques Used: Inhibition

    CK2 inhibition reduces bursting in CA1. (A) Sample traces of CA1 pyramidal cell intracellular recordings from a vehicle-pretreated epileptic rat (Pilo, black traces) and a TBB-pretreated epileptic rat (Pilo-TBB, blue traces). Note the bursting of the upper traces upon just suprathreshold stimulation. (B) Proportions of burster and non-burster neurons in the four experimental groups (* P < 0.05). (C,D) Afterdepolarizing potential (ADP) with different pulse durations ( d = 3, 5, 7 ms). The area under curve of the ADP (starting from d + 5 ms, indicated in gray in C ) of cells from vehicle-pretreated epileptic animals (Pilo) was significant against the ADP of all other experimental groups (* P < 0.05, three-way ANOVA with Tukey post hoc test).
    Figure Legend Snippet: CK2 inhibition reduces bursting in CA1. (A) Sample traces of CA1 pyramidal cell intracellular recordings from a vehicle-pretreated epileptic rat (Pilo, black traces) and a TBB-pretreated epileptic rat (Pilo-TBB, blue traces). Note the bursting of the upper traces upon just suprathreshold stimulation. (B) Proportions of burster and non-burster neurons in the four experimental groups (* P < 0.05). (C,D) Afterdepolarizing potential (ADP) with different pulse durations ( d = 3, 5, 7 ms). The area under curve of the ADP (starting from d + 5 ms, indicated in gray in C ) of cells from vehicle-pretreated epileptic animals (Pilo) was significant against the ADP of all other experimental groups (* P < 0.05, three-way ANOVA with Tukey post hoc test).

    Techniques Used: Inhibition

    CK2 inhibition modifies KC2 function. (A) Bar graph of the mAHP-mediating current in all four experimental groups. CA1 cells from vehicle-pretreated epileptic animals (Pilo) showed significantly less mAHP currents than control cells and cells from TBB-pretreated epileptic animals. (B) The sAHP charge transfer is significantly smaller in epileptic animals than in controls. TBB has no effect on the sAHP charge transfer. (C) Quantitative RT-PCR analysis of the CA1 subfield reveals that tissue from TBB-pretreated control animals contained significantly more K Ca 2.2 mRNA than vehicle-pretreated controls and TBB-pretreated epileptic rats. (D) Western blot analysis of CK2 protein in TBB-pretreated tissue. Note the region-specific differences between control and epileptic tissues. * P < 0.05.
    Figure Legend Snippet: CK2 inhibition modifies KC2 function. (A) Bar graph of the mAHP-mediating current in all four experimental groups. CA1 cells from vehicle-pretreated epileptic animals (Pilo) showed significantly less mAHP currents than control cells and cells from TBB-pretreated epileptic animals. (B) The sAHP charge transfer is significantly smaller in epileptic animals than in controls. TBB has no effect on the sAHP charge transfer. (C) Quantitative RT-PCR analysis of the CA1 subfield reveals that tissue from TBB-pretreated control animals contained significantly more K Ca 2.2 mRNA than vehicle-pretreated controls and TBB-pretreated epileptic rats. (D) Western blot analysis of CK2 protein in TBB-pretreated tissue. Note the region-specific differences between control and epileptic tissues. * P < 0.05.

    Techniques Used: Inhibition, Quantitative RT-PCR, Western Blot

    CK2 inhibition modifies HCN channel function. (A) Sample traces of the membrane potential following hyperpolarizing and depolarizing pulse steps in intracellular recordings from CA1 neurons (gray: control, black: vehicle-pretreated epileptic, blue: TBB-pretreated epileptic). (B) Bar graphs of the train-AHP amplitude and voltage sag obtained from recordings shown in A . The train-AHP was significantly smaller in epileptic tissues regardless of the treatment with TBB. In contrast, the voltage sag was enhanced in TBB-pretreated tissue from both control and epileptic animals. Quantitative RT-PCR analysis of the CA1 subfield of all four HCN channel isoforms revealed significant and inverse expression changes for HCN1 (C) and HCN3 (E) , but no changes for HCN2 (D) and HCN4 (F) . * P < 0.05.
    Figure Legend Snippet: CK2 inhibition modifies HCN channel function. (A) Sample traces of the membrane potential following hyperpolarizing and depolarizing pulse steps in intracellular recordings from CA1 neurons (gray: control, black: vehicle-pretreated epileptic, blue: TBB-pretreated epileptic). (B) Bar graphs of the train-AHP amplitude and voltage sag obtained from recordings shown in A . The train-AHP was significantly smaller in epileptic tissues regardless of the treatment with TBB. In contrast, the voltage sag was enhanced in TBB-pretreated tissue from both control and epileptic animals. Quantitative RT-PCR analysis of the CA1 subfield of all four HCN channel isoforms revealed significant and inverse expression changes for HCN1 (C) and HCN3 (E) , but no changes for HCN2 (D) and HCN4 (F) . * P < 0.05.

    Techniques Used: Inhibition, Quantitative RT-PCR, Expressing

    CK2 inhibition enhances train-AHP and reduces excitability in HCN-blocking conditions. (A) Proportions of burster and non-burster neurons in the four experimental groups in the presence of the HCN channel blocker ZD7288. (B) Sample traces of intracellular recordings following prolonged depolarization (600 ms) used for train-AHP amplitude (e.g., see blue arrow) and number of action potentials. For comparison, sample traces in the absence of ZD7288 were also given (left side). (C) Bar graph of the train-AHP amplitude shows a significantly increased train-AHP in cells from TBB-pretreated epileptic rats (blue bar graph). For the sake of clarity, the train-AHP in the absence of ZD7288 (gray bar graphs, values from ) are also shown. Significant effects were detected between animal groups (Ctrl versus Pilo, * P < 0.05) and pretreatment groups (vehicle versus TBB, * P < 0.05), but not for the ZD7288 effect by three-way ANOVA with Holm–Sidak post hoc tests. However, there was a significant interaction between ZD7288 and pretreatment with TBB or vehicle (* P < 0.05). (D) Number of action potentials during 600 ms depolarization (from 100 to 500 pA). Two-way ANOVA with Tukey post hoc tests showed a trend effect between animal groups (Ctrl versus Pilo, + P = 0.08) and a significant effect treatment groups (vehicle versus TBB, * P < 0.05).
    Figure Legend Snippet: CK2 inhibition enhances train-AHP and reduces excitability in HCN-blocking conditions. (A) Proportions of burster and non-burster neurons in the four experimental groups in the presence of the HCN channel blocker ZD7288. (B) Sample traces of intracellular recordings following prolonged depolarization (600 ms) used for train-AHP amplitude (e.g., see blue arrow) and number of action potentials. For comparison, sample traces in the absence of ZD7288 were also given (left side). (C) Bar graph of the train-AHP amplitude shows a significantly increased train-AHP in cells from TBB-pretreated epileptic rats (blue bar graph). For the sake of clarity, the train-AHP in the absence of ZD7288 (gray bar graphs, values from ) are also shown. Significant effects were detected between animal groups (Ctrl versus Pilo, * P < 0.05) and pretreatment groups (vehicle versus TBB, * P < 0.05), but not for the ZD7288 effect by three-way ANOVA with Holm–Sidak post hoc tests. However, there was a significant interaction between ZD7288 and pretreatment with TBB or vehicle (* P < 0.05). (D) Number of action potentials during 600 ms depolarization (from 100 to 500 pA). Two-way ANOVA with Tukey post hoc tests showed a trend effect between animal groups (Ctrl versus Pilo, + P = 0.08) and a significant effect treatment groups (vehicle versus TBB, * P < 0.05).

    Techniques Used: Inhibition, Blocking Assay

    ck2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ck2
    Ck2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti phospho ck2 substrate antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho ck2 substrate antibody
    Anti Phospho Ck2 Substrate Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ck2 phosphosubstrate  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ck2 phosphosubstrate
    Ck2 Phosphosubstrate, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti phospho ck2 substrate  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho ck2 substrate
    <t>CK2</t> kinase activity is essential for Drosophila oogenesis. A, identification of novel kinase regulators of Drosophila oogenesis. The indicated 78 kinases (on the x axis) were knocked down in the female germ line using nanos-Gal4:VP16-driven shRNA. Shown are box plots depicting egg laying rates of these flies (n = 20). Bars indicate the first to third quartiles, horizontal black lines denote the median, and circles denote outliers. Red bars indicate kinases whose reduced expression resulted in decreased numbers of eggs laid, whereas green bars indicate kinases whose reduced expression caused an increase in the number of eggs laid. The blue bar denotes the egg laying rate of flies expressing a control shRNA. Whiskers represent the upper and lower limits of the range. Statistical significance was determined using a Mann-Whitney U test. B, reduced ovarian size upon reduction of CK2 expression. Ovaries from flies of the indicated genotypes were dissected and imaged by light microscopy. Scale bar, 1 mm. C, egg laying rates from flies of the indicated genotypes. Expression of shRNA and/or transgenes was driven using either nanos-Gal4 (green) or matα-Gal4 (blue). Data are presented as in A. D, transgenic expression of epitope-tagged human CK2α and CK2β in the Drosophila female germ line. Ovary lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. Note that CK2α-HA migrates as a doublet.
    Anti Phospho Ck2 Substrate, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis"

    Article Title: The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.814657

    CK2 kinase activity is essential for Drosophila oogenesis. A, identification of novel kinase regulators of Drosophila oogenesis. The indicated 78 kinases (on the x axis) were knocked down in the female germ line using nanos-Gal4:VP16-driven shRNA. Shown are box plots depicting egg laying rates of these flies (n = 20). Bars indicate the first to third quartiles, horizontal black lines denote the median, and circles denote outliers. Red bars indicate kinases whose reduced expression resulted in decreased numbers of eggs laid, whereas green bars indicate kinases whose reduced expression caused an increase in the number of eggs laid. The blue bar denotes the egg laying rate of flies expressing a control shRNA. Whiskers represent the upper and lower limits of the range. Statistical significance was determined using a Mann-Whitney U test. B, reduced ovarian size upon reduction of CK2 expression. Ovaries from flies of the indicated genotypes were dissected and imaged by light microscopy. Scale bar, 1 mm. C, egg laying rates from flies of the indicated genotypes. Expression of shRNA and/or transgenes was driven using either nanos-Gal4 (green) or matα-Gal4 (blue). Data are presented as in A. D, transgenic expression of epitope-tagged human CK2α and CK2β in the Drosophila female germ line. Ovary lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. Note that CK2α-HA migrates as a doublet.
    Figure Legend Snippet: CK2 kinase activity is essential for Drosophila oogenesis. A, identification of novel kinase regulators of Drosophila oogenesis. The indicated 78 kinases (on the x axis) were knocked down in the female germ line using nanos-Gal4:VP16-driven shRNA. Shown are box plots depicting egg laying rates of these flies (n = 20). Bars indicate the first to third quartiles, horizontal black lines denote the median, and circles denote outliers. Red bars indicate kinases whose reduced expression resulted in decreased numbers of eggs laid, whereas green bars indicate kinases whose reduced expression caused an increase in the number of eggs laid. The blue bar denotes the egg laying rate of flies expressing a control shRNA. Whiskers represent the upper and lower limits of the range. Statistical significance was determined using a Mann-Whitney U test. B, reduced ovarian size upon reduction of CK2 expression. Ovaries from flies of the indicated genotypes were dissected and imaged by light microscopy. Scale bar, 1 mm. C, egg laying rates from flies of the indicated genotypes. Expression of shRNA and/or transgenes was driven using either nanos-Gal4 (green) or matα-Gal4 (blue). Data are presented as in A. D, transgenic expression of epitope-tagged human CK2α and CK2β in the Drosophila female germ line. Ovary lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. Note that CK2α-HA migrates as a doublet.

    Techniques Used: Activity Assay, shRNA, Expressing, MANN-WHITNEY, Light Microscopy, Transgenic Assay, SDS Page

    A biochemical screen identifies substrates of CK2 in the Drosophila ovary. A, schematic of the strategy used to identify novel ovarian substrates of CK2. B, experimental validation of the approach outlined in A. Ovary lysate was incubated for 30 min at 30 °C with or without recombinant human CK2 holoenzyme and in the presence or absence of CX-4945 (100 μm) as indicated. GTPγS (2.5 mm) was then added, and the reactions were incubated further for 1 min at 30 °C followed by treatment with PNBM for 60 min at room temperature. Kinase reactions were subsequently resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, immunoprecipitation (IP) of putative CK2 substrates from Drosophila ovary lysate. An anti-thiophosphate ester antibody was used to immunoprecipitate thiophosphorylated proteins. Immunoprecipitated proteins were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. The yellow asterisk indicates the position of the band that was excised from a corresponding Coomassie Blue–stained gel for protein identification by LC-MS/MS. D, primary amino acid sequence of Jabba (isoform F). Highlighted in red are unique peptides, representing 7% coverage of the protein, identified by mass spectrometry.
    Figure Legend Snippet: A biochemical screen identifies substrates of CK2 in the Drosophila ovary. A, schematic of the strategy used to identify novel ovarian substrates of CK2. B, experimental validation of the approach outlined in A. Ovary lysate was incubated for 30 min at 30 °C with or without recombinant human CK2 holoenzyme and in the presence or absence of CX-4945 (100 μm) as indicated. GTPγS (2.5 mm) was then added, and the reactions were incubated further for 1 min at 30 °C followed by treatment with PNBM for 60 min at room temperature. Kinase reactions were subsequently resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, immunoprecipitation (IP) of putative CK2 substrates from Drosophila ovary lysate. An anti-thiophosphate ester antibody was used to immunoprecipitate thiophosphorylated proteins. Immunoprecipitated proteins were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. The yellow asterisk indicates the position of the band that was excised from a corresponding Coomassie Blue–stained gel for protein identification by LC-MS/MS. D, primary amino acid sequence of Jabba (isoform F). Highlighted in red are unique peptides, representing 7% coverage of the protein, identified by mass spectrometry.

    Techniques Used: Incubation, Recombinant, SDS Page, Immunoprecipitation, Western Blot, Staining, Liquid Chromatography with Mass Spectroscopy, Sequencing, Mass Spectrometry

    Jabba is a novel CK2 substrate. A, in vitro kinase assay using GST-Jabba and CK2. GST-Jabba (or no substrate) was incubated in the presence or absence of recombinant CK2 holoenzyme and ATPγS followed by treatment with PNBM. Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. B, inhibition of CK2 kinase activity using the small-molecule inhibitor CX-4945. HEK293 cells were treated (or not) with the indicated concentrations of CX-4945 for 24 h. Cell lysates (40 μg of total protein) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, HEK293 cells were transfected (or not) with a plasmid encoding GFP-Jabba. Cell lysates (5 μg of total protein) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. D, HEK293 cells were cotransfected with plasmids encoding GFP-Jabba and pCMV-HA (empty vector) or pCMV-CK2α-HA. Cells were lysed followed by immunoprecipitation of GFP-Jabba and immunoblotting of the precipitated protein with the indicated antibodies. Western blots shown are representative of three independent experiments. IP, immunoprecipitate; WCL, whole cell lysate.
    Figure Legend Snippet: Jabba is a novel CK2 substrate. A, in vitro kinase assay using GST-Jabba and CK2. GST-Jabba (or no substrate) was incubated in the presence or absence of recombinant CK2 holoenzyme and ATPγS followed by treatment with PNBM. Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. B, inhibition of CK2 kinase activity using the small-molecule inhibitor CX-4945. HEK293 cells were treated (or not) with the indicated concentrations of CX-4945 for 24 h. Cell lysates (40 μg of total protein) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, HEK293 cells were transfected (or not) with a plasmid encoding GFP-Jabba. Cell lysates (5 μg of total protein) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. D, HEK293 cells were cotransfected with plasmids encoding GFP-Jabba and pCMV-HA (empty vector) or pCMV-CK2α-HA. Cells were lysed followed by immunoprecipitation of GFP-Jabba and immunoblotting of the precipitated protein with the indicated antibodies. Western blots shown are representative of three independent experiments. IP, immunoprecipitate; WCL, whole cell lysate.

    Techniques Used: In Vitro, Kinase Assay, Incubation, Recombinant, SDS Page, Western Blot, Inhibition, Activity Assay, Transfection, Plasmid Preparation, Immunoprecipitation

    CK2 phosphorylates multiple regions of Jabba. A, schematic diagram of Jabba (isoform F) depicting various truncation fragments generated. B, in vitro kinase assay using GST-Jabba(222–273) and CK2. GST or GST-Jabba(222–273) was incubated with recombinant CK2 holoenzyme and ATPγS in the presence or absence of CX-4945 (10 μm) followed by treatment with PNBM. Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. C, in vitro kinase assay using GST-Jabba(274–537) and CK2. GST or GST-Jabba(274–537) was incubated with recombinant CK2 holoenzyme and ATP in the presence or absence of CX-4945 (10 μm). Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments.
    Figure Legend Snippet: CK2 phosphorylates multiple regions of Jabba. A, schematic diagram of Jabba (isoform F) depicting various truncation fragments generated. B, in vitro kinase assay using GST-Jabba(222–273) and CK2. GST or GST-Jabba(222–273) was incubated with recombinant CK2 holoenzyme and ATPγS in the presence or absence of CX-4945 (10 μm) followed by treatment with PNBM. Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. C, in vitro kinase assay using GST-Jabba(274–537) and CK2. GST or GST-Jabba(274–537) was incubated with recombinant CK2 holoenzyme and ATP in the presence or absence of CX-4945 (10 μm). Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments.

    Techniques Used: Generated, In Vitro, Kinase Assay, Incubation, Recombinant, SDS Page, Western Blot

    Jabba localizes to lipid droplets in a CK2-independent manner. A, 3T3-L1 preadipocytes were cotransfected with plasmids encoding myc-perilipin-3 (Plin-3) and GFP. Cells were treated with oleic acid. Cells were then treated with DMSO (top panels) or CX-4945 (10 μm) (bottom panels), fixed, and processed for immunofluorescence microscopy with anti-myc antibodies. Cells were counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm. B, 3T3-L1 preadipocytes were cotransfected with plasmids encoding myc-perlipin-3 (Plin-3) and GFP-Jabba and incubated with oleic acid. Cells were then treated with DMSO (top panels) or CX-4945 (10 μm) (bottom panels), fixed, and processed for immunofluorescence microscopy with anti-myc antibody. Cells were counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm.
    Figure Legend Snippet: Jabba localizes to lipid droplets in a CK2-independent manner. A, 3T3-L1 preadipocytes were cotransfected with plasmids encoding myc-perilipin-3 (Plin-3) and GFP. Cells were treated with oleic acid. Cells were then treated with DMSO (top panels) or CX-4945 (10 μm) (bottom panels), fixed, and processed for immunofluorescence microscopy with anti-myc antibodies. Cells were counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm. B, 3T3-L1 preadipocytes were cotransfected with plasmids encoding myc-perlipin-3 (Plin-3) and GFP-Jabba and incubated with oleic acid. Cells were then treated with DMSO (top panels) or CX-4945 (10 μm) (bottom panels), fixed, and processed for immunofluorescence microscopy with anti-myc antibody. Cells were counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm.

    Techniques Used: Immunofluorescence, Microscopy, Incubation

    Jabba and CK2 regulate lipid metabolism during Drosophila oogenesis. A, ovaries from flies of the indicated genotypes were dissected and stained with Oil Red O for visualization of neutral lipids. Shown are representative stage 10 egg chambers. Scale bar, 100 μm. B, quantitation of total triglyceride levels from ovaries of flies of the indicated genotypes. Data are presented as the mean (n = 4) with error bars denoting S.D. * indicates a p value <0.05. ns, not significant. C, confocal immunofluorescence microscopy of egg chambers from flies of the indicated genotypes. Dissected egg chambers were fixed and stained with anti-myc antibody to detect Jabba-labeled lipid droplets (green) and counterstained with rhodamine phalloidin to label F-actin (red). Shown are single optical confocal sections. Scale bars, 50 μm.
    Figure Legend Snippet: Jabba and CK2 regulate lipid metabolism during Drosophila oogenesis. A, ovaries from flies of the indicated genotypes were dissected and stained with Oil Red O for visualization of neutral lipids. Shown are representative stage 10 egg chambers. Scale bar, 100 μm. B, quantitation of total triglyceride levels from ovaries of flies of the indicated genotypes. Data are presented as the mean (n = 4) with error bars denoting S.D. * indicates a p value <0.05. ns, not significant. C, confocal immunofluorescence microscopy of egg chambers from flies of the indicated genotypes. Dissected egg chambers were fixed and stained with anti-myc antibody to detect Jabba-labeled lipid droplets (green) and counterstained with rhodamine phalloidin to label F-actin (red). Shown are single optical confocal sections. Scale bars, 50 μm.

    Techniques Used: Staining, Quantitation Assay, Immunofluorescence, Microscopy, Labeling

    anti phospho ck2 substrate  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho ck2 substrate
    <t>CK2</t> kinase activity is essential for Drosophila oogenesis. A, identification of novel kinase regulators of Drosophila oogenesis. The indicated 78 kinases (on the x axis) were knocked down in the female germ line using nanos-Gal4:VP16-driven shRNA. Shown are box plots depicting egg laying rates of these flies (n = 20). Bars indicate the first to third quartiles, horizontal black lines denote the median, and circles denote outliers. Red bars indicate kinases whose reduced expression resulted in decreased numbers of eggs laid, whereas green bars indicate kinases whose reduced expression caused an increase in the number of eggs laid. The blue bar denotes the egg laying rate of flies expressing a control shRNA. Whiskers represent the upper and lower limits of the range. Statistical significance was determined using a Mann-Whitney U test. B, reduced ovarian size upon reduction of CK2 expression. Ovaries from flies of the indicated genotypes were dissected and imaged by light microscopy. Scale bar, 1 mm. C, egg laying rates from flies of the indicated genotypes. Expression of shRNA and/or transgenes was driven using either nanos-Gal4 (green) or matα-Gal4 (blue). Data are presented as in A. D, transgenic expression of epitope-tagged human CK2α and CK2β in the Drosophila female germ line. Ovary lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. Note that CK2α-HA migrates as a doublet.
    Anti Phospho Ck2 Substrate, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho ck2 substrate/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti phospho ck2 substrate - by Bioz Stars, 2023-02
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    Images

    1) Product Images from "The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis"

    Article Title: The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.814657

    CK2 kinase activity is essential for Drosophila oogenesis. A, identification of novel kinase regulators of Drosophila oogenesis. The indicated 78 kinases (on the x axis) were knocked down in the female germ line using nanos-Gal4:VP16-driven shRNA. Shown are box plots depicting egg laying rates of these flies (n = 20). Bars indicate the first to third quartiles, horizontal black lines denote the median, and circles denote outliers. Red bars indicate kinases whose reduced expression resulted in decreased numbers of eggs laid, whereas green bars indicate kinases whose reduced expression caused an increase in the number of eggs laid. The blue bar denotes the egg laying rate of flies expressing a control shRNA. Whiskers represent the upper and lower limits of the range. Statistical significance was determined using a Mann-Whitney U test. B, reduced ovarian size upon reduction of CK2 expression. Ovaries from flies of the indicated genotypes were dissected and imaged by light microscopy. Scale bar, 1 mm. C, egg laying rates from flies of the indicated genotypes. Expression of shRNA and/or transgenes was driven using either nanos-Gal4 (green) or matα-Gal4 (blue). Data are presented as in A. D, transgenic expression of epitope-tagged human CK2α and CK2β in the Drosophila female germ line. Ovary lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. Note that CK2α-HA migrates as a doublet.
    Figure Legend Snippet: CK2 kinase activity is essential for Drosophila oogenesis. A, identification of novel kinase regulators of Drosophila oogenesis. The indicated 78 kinases (on the x axis) were knocked down in the female germ line using nanos-Gal4:VP16-driven shRNA. Shown are box plots depicting egg laying rates of these flies (n = 20). Bars indicate the first to third quartiles, horizontal black lines denote the median, and circles denote outliers. Red bars indicate kinases whose reduced expression resulted in decreased numbers of eggs laid, whereas green bars indicate kinases whose reduced expression caused an increase in the number of eggs laid. The blue bar denotes the egg laying rate of flies expressing a control shRNA. Whiskers represent the upper and lower limits of the range. Statistical significance was determined using a Mann-Whitney U test. B, reduced ovarian size upon reduction of CK2 expression. Ovaries from flies of the indicated genotypes were dissected and imaged by light microscopy. Scale bar, 1 mm. C, egg laying rates from flies of the indicated genotypes. Expression of shRNA and/or transgenes was driven using either nanos-Gal4 (green) or matα-Gal4 (blue). Data are presented as in A. D, transgenic expression of epitope-tagged human CK2α and CK2β in the Drosophila female germ line. Ovary lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. Note that CK2α-HA migrates as a doublet.

    Techniques Used: Activity Assay, shRNA, Expressing, MANN-WHITNEY, Light Microscopy, Transgenic Assay, SDS Page

    A biochemical screen identifies substrates of CK2 in the Drosophila ovary. A, schematic of the strategy used to identify novel ovarian substrates of CK2. B, experimental validation of the approach outlined in A. Ovary lysate was incubated for 30 min at 30 °C with or without recombinant human CK2 holoenzyme and in the presence or absence of CX-4945 (100 μm) as indicated. GTPγS (2.5 mm) was then added, and the reactions were incubated further for 1 min at 30 °C followed by treatment with PNBM for 60 min at room temperature. Kinase reactions were subsequently resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, immunoprecipitation (IP) of putative CK2 substrates from Drosophila ovary lysate. An anti-thiophosphate ester antibody was used to immunoprecipitate thiophosphorylated proteins. Immunoprecipitated proteins were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. The yellow asterisk indicates the position of the band that was excised from a corresponding Coomassie Blue–stained gel for protein identification by LC-MS/MS. D, primary amino acid sequence of Jabba (isoform F). Highlighted in red are unique peptides, representing 7% coverage of the protein, identified by mass spectrometry.
    Figure Legend Snippet: A biochemical screen identifies substrates of CK2 in the Drosophila ovary. A, schematic of the strategy used to identify novel ovarian substrates of CK2. B, experimental validation of the approach outlined in A. Ovary lysate was incubated for 30 min at 30 °C with or without recombinant human CK2 holoenzyme and in the presence or absence of CX-4945 (100 μm) as indicated. GTPγS (2.5 mm) was then added, and the reactions were incubated further for 1 min at 30 °C followed by treatment with PNBM for 60 min at room temperature. Kinase reactions were subsequently resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, immunoprecipitation (IP) of putative CK2 substrates from Drosophila ovary lysate. An anti-thiophosphate ester antibody was used to immunoprecipitate thiophosphorylated proteins. Immunoprecipitated proteins were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. The yellow asterisk indicates the position of the band that was excised from a corresponding Coomassie Blue–stained gel for protein identification by LC-MS/MS. D, primary amino acid sequence of Jabba (isoform F). Highlighted in red are unique peptides, representing 7% coverage of the protein, identified by mass spectrometry.

    Techniques Used: Incubation, Recombinant, SDS Page, Immunoprecipitation, Western Blot, Staining, Liquid Chromatography with Mass Spectroscopy, Sequencing, Mass Spectrometry

    Jabba is a novel CK2 substrate. A, in vitro kinase assay using GST-Jabba and CK2. GST-Jabba (or no substrate) was incubated in the presence or absence of recombinant CK2 holoenzyme and ATPγS followed by treatment with PNBM. Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. B, inhibition of CK2 kinase activity using the small-molecule inhibitor CX-4945. HEK293 cells were treated (or not) with the indicated concentrations of CX-4945 for 24 h. Cell lysates (40 μg of total protein) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, HEK293 cells were transfected (or not) with a plasmid encoding GFP-Jabba. Cell lysates (5 μg of total protein) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. D, HEK293 cells were cotransfected with plasmids encoding GFP-Jabba and pCMV-HA (empty vector) or pCMV-CK2α-HA. Cells were lysed followed by immunoprecipitation of GFP-Jabba and immunoblotting of the precipitated protein with the indicated antibodies. Western blots shown are representative of three independent experiments. IP, immunoprecipitate; WCL, whole cell lysate.
    Figure Legend Snippet: Jabba is a novel CK2 substrate. A, in vitro kinase assay using GST-Jabba and CK2. GST-Jabba (or no substrate) was incubated in the presence or absence of recombinant CK2 holoenzyme and ATPγS followed by treatment with PNBM. Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. B, inhibition of CK2 kinase activity using the small-molecule inhibitor CX-4945. HEK293 cells were treated (or not) with the indicated concentrations of CX-4945 for 24 h. Cell lysates (40 μg of total protein) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, HEK293 cells were transfected (or not) with a plasmid encoding GFP-Jabba. Cell lysates (5 μg of total protein) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. D, HEK293 cells were cotransfected with plasmids encoding GFP-Jabba and pCMV-HA (empty vector) or pCMV-CK2α-HA. Cells were lysed followed by immunoprecipitation of GFP-Jabba and immunoblotting of the precipitated protein with the indicated antibodies. Western blots shown are representative of three independent experiments. IP, immunoprecipitate; WCL, whole cell lysate.

    Techniques Used: In Vitro, Kinase Assay, Incubation, Recombinant, SDS Page, Western Blot, Inhibition, Activity Assay, Transfection, Plasmid Preparation, Immunoprecipitation

    CK2 phosphorylates multiple regions of Jabba. A, schematic diagram of Jabba (isoform F) depicting various truncation fragments generated. B, in vitro kinase assay using GST-Jabba(222–273) and CK2. GST or GST-Jabba(222–273) was incubated with recombinant CK2 holoenzyme and ATPγS in the presence or absence of CX-4945 (10 μm) followed by treatment with PNBM. Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. C, in vitro kinase assay using GST-Jabba(274–537) and CK2. GST or GST-Jabba(274–537) was incubated with recombinant CK2 holoenzyme and ATP in the presence or absence of CX-4945 (10 μm). Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments.
    Figure Legend Snippet: CK2 phosphorylates multiple regions of Jabba. A, schematic diagram of Jabba (isoform F) depicting various truncation fragments generated. B, in vitro kinase assay using GST-Jabba(222–273) and CK2. GST or GST-Jabba(222–273) was incubated with recombinant CK2 holoenzyme and ATPγS in the presence or absence of CX-4945 (10 μm) followed by treatment with PNBM. Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. C, in vitro kinase assay using GST-Jabba(274–537) and CK2. GST or GST-Jabba(274–537) was incubated with recombinant CK2 holoenzyme and ATP in the presence or absence of CX-4945 (10 μm). Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments.

    Techniques Used: Generated, In Vitro, Kinase Assay, Incubation, Recombinant, SDS Page, Western Blot

    Jabba localizes to lipid droplets in a CK2-independent manner. A, 3T3-L1 preadipocytes were cotransfected with plasmids encoding myc-perilipin-3 (Plin-3) and GFP. Cells were treated with oleic acid. Cells were then treated with DMSO (top panels) or CX-4945 (10 μm) (bottom panels), fixed, and processed for immunofluorescence microscopy with anti-myc antibodies. Cells were counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm. B, 3T3-L1 preadipocytes were cotransfected with plasmids encoding myc-perlipin-3 (Plin-3) and GFP-Jabba and incubated with oleic acid. Cells were then treated with DMSO (top panels) or CX-4945 (10 μm) (bottom panels), fixed, and processed for immunofluorescence microscopy with anti-myc antibody. Cells were counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm.
    Figure Legend Snippet: Jabba localizes to lipid droplets in a CK2-independent manner. A, 3T3-L1 preadipocytes were cotransfected with plasmids encoding myc-perilipin-3 (Plin-3) and GFP. Cells were treated with oleic acid. Cells were then treated with DMSO (top panels) or CX-4945 (10 μm) (bottom panels), fixed, and processed for immunofluorescence microscopy with anti-myc antibodies. Cells were counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm. B, 3T3-L1 preadipocytes were cotransfected with plasmids encoding myc-perlipin-3 (Plin-3) and GFP-Jabba and incubated with oleic acid. Cells were then treated with DMSO (top panels) or CX-4945 (10 μm) (bottom panels), fixed, and processed for immunofluorescence microscopy with anti-myc antibody. Cells were counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm.

    Techniques Used: Immunofluorescence, Microscopy, Incubation

    Jabba and CK2 regulate lipid metabolism during Drosophila oogenesis. A, ovaries from flies of the indicated genotypes were dissected and stained with Oil Red O for visualization of neutral lipids. Shown are representative stage 10 egg chambers. Scale bar, 100 μm. B, quantitation of total triglyceride levels from ovaries of flies of the indicated genotypes. Data are presented as the mean (n = 4) with error bars denoting S.D. * indicates a p value <0.05. ns, not significant. C, confocal immunofluorescence microscopy of egg chambers from flies of the indicated genotypes. Dissected egg chambers were fixed and stained with anti-myc antibody to detect Jabba-labeled lipid droplets (green) and counterstained with rhodamine phalloidin to label F-actin (red). Shown are single optical confocal sections. Scale bars, 50 μm.
    Figure Legend Snippet: Jabba and CK2 regulate lipid metabolism during Drosophila oogenesis. A, ovaries from flies of the indicated genotypes were dissected and stained with Oil Red O for visualization of neutral lipids. Shown are representative stage 10 egg chambers. Scale bar, 100 μm. B, quantitation of total triglyceride levels from ovaries of flies of the indicated genotypes. Data are presented as the mean (n = 4) with error bars denoting S.D. * indicates a p value <0.05. ns, not significant. C, confocal immunofluorescence microscopy of egg chambers from flies of the indicated genotypes. Dissected egg chambers were fixed and stained with anti-myc antibody to detect Jabba-labeled lipid droplets (green) and counterstained with rhodamine phalloidin to label F-actin (red). Shown are single optical confocal sections. Scale bars, 50 μm.

    Techniques Used: Staining, Quantitation Assay, Immunofluorescence, Microscopy, Labeling

    anti phospho ck2 substrate  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho ck2 substrate
    Anti Phospho Ck2 Substrate, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho ck2 substrate
    Proteins Co-Immunoprecipitating With NOXO1 in T84 Cells Stimulated by TNFα (5 ng/mL) + IL17 (50 ng/mL) and Related to NADPH Oxidase or Redox Signaling
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    Cell Signaling Technology Inc phospho ser thr ck2 substrate rabbit
    List of commercial primary antibodies used for Western blot.
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    <t>CK2</t> inhibition exerts disease modifying properties. (A) Time frame of the experiment. TBB was administered four times in vivo on days “−3” (i.e., 3 days before status epilepticus) through “0” (i.e., day of pilocarpine-induced status epilepticus). All experiments thereafter were performed during the chronic stage of recurrent seizures. (B–D) Latency to the first stage 4 or 5 seizure after pilocarpine injection in vehicle-pretreated (B) and TBB-pretreated (C) rats. The median latency was significantly (*** P < 0.001, Mann-Whitney U-test) prolonged in the TBB-pretreated group (D) . (E) Number of interictal spikes per hour (left y -axis) in the electroencephalogram (EEG) during 3 days (97–100 post-SE). Note that the number of daily seizures during this period is also depicted (diamonds, right y -axis). There was a significant group effect of TBB pretreatment (** P < 0.01, two-way ANOVA with Tukey post hoc test). (F) Number of generalized seizures (at least stage 3) at daylight (from 06:00 to 18:00 h) for both animal groups as grand average for the entire recording period. (G) Seizure burden progression as fold change of seizure rates during epileptogenesis. The variability was significantly reduced in TBB-pretreated epileptic animals (** P < 0.01, F -test). (H) Relative risk reduction (RRR) of seizure progression by TBB pretreatment was significant (* P < 0.05, t -test).
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    <t>CK2</t> inhibition exerts disease modifying properties. (A) Time frame of the experiment. TBB was administered four times in vivo on days “−3” (i.e., 3 days before status epilepticus) through “0” (i.e., day of pilocarpine-induced status epilepticus). All experiments thereafter were performed during the chronic stage of recurrent seizures. (B–D) Latency to the first stage 4 or 5 seizure after pilocarpine injection in vehicle-pretreated (B) and TBB-pretreated (C) rats. The median latency was significantly (*** P < 0.001, Mann-Whitney U-test) prolonged in the TBB-pretreated group (D) . (E) Number of interictal spikes per hour (left y -axis) in the electroencephalogram (EEG) during 3 days (97–100 post-SE). Note that the number of daily seizures during this period is also depicted (diamonds, right y -axis). There was a significant group effect of TBB pretreatment (** P < 0.01, two-way ANOVA with Tukey post hoc test). (F) Number of generalized seizures (at least stage 3) at daylight (from 06:00 to 18:00 h) for both animal groups as grand average for the entire recording period. (G) Seizure burden progression as fold change of seizure rates during epileptogenesis. The variability was significantly reduced in TBB-pretreated epileptic animals (** P < 0.01, F -test). (H) Relative risk reduction (RRR) of seizure progression by TBB pretreatment was significant (* P < 0.05, t -test).
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    <t>CK2</t> inhibition exerts disease modifying properties. (A) Time frame of the experiment. TBB was administered four times in vivo on days “−3” (i.e., 3 days before status epilepticus) through “0” (i.e., day of pilocarpine-induced status epilepticus). All experiments thereafter were performed during the chronic stage of recurrent seizures. (B–D) Latency to the first stage 4 or 5 seizure after pilocarpine injection in vehicle-pretreated (B) and TBB-pretreated (C) rats. The median latency was significantly (*** P < 0.001, Mann-Whitney U-test) prolonged in the TBB-pretreated group (D) . (E) Number of interictal spikes per hour (left y -axis) in the electroencephalogram (EEG) during 3 days (97–100 post-SE). Note that the number of daily seizures during this period is also depicted (diamonds, right y -axis). There was a significant group effect of TBB pretreatment (** P < 0.01, two-way ANOVA with Tukey post hoc test). (F) Number of generalized seizures (at least stage 3) at daylight (from 06:00 to 18:00 h) for both animal groups as grand average for the entire recording period. (G) Seizure burden progression as fold change of seizure rates during epileptogenesis. The variability was significantly reduced in TBB-pretreated epileptic animals (** P < 0.01, F -test). (H) Relative risk reduction (RRR) of seizure progression by TBB pretreatment was significant (* P < 0.05, t -test).
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    Cell Signaling Technology Inc ck2 phosphosubstrate
    <t>CK2</t> inhibition exerts disease modifying properties. (A) Time frame of the experiment. TBB was administered four times in vivo on days “−3” (i.e., 3 days before status epilepticus) through “0” (i.e., day of pilocarpine-induced status epilepticus). All experiments thereafter were performed during the chronic stage of recurrent seizures. (B–D) Latency to the first stage 4 or 5 seizure after pilocarpine injection in vehicle-pretreated (B) and TBB-pretreated (C) rats. The median latency was significantly (*** P < 0.001, Mann-Whitney U-test) prolonged in the TBB-pretreated group (D) . (E) Number of interictal spikes per hour (left y -axis) in the electroencephalogram (EEG) during 3 days (97–100 post-SE). Note that the number of daily seizures during this period is also depicted (diamonds, right y -axis). There was a significant group effect of TBB pretreatment (** P < 0.01, two-way ANOVA with Tukey post hoc test). (F) Number of generalized seizures (at least stage 3) at daylight (from 06:00 to 18:00 h) for both animal groups as grand average for the entire recording period. (G) Seizure burden progression as fold change of seizure rates during epileptogenesis. The variability was significantly reduced in TBB-pretreated epileptic animals (** P < 0.01, F -test). (H) Relative risk reduction (RRR) of seizure progression by TBB pretreatment was significant (* P < 0.05, t -test).
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    Cell Signaling Technology Inc anti phospho ck2 substrate
    <t>CK2</t> kinase activity is essential for Drosophila oogenesis. A, identification of novel kinase regulators of Drosophila oogenesis. The indicated 78 kinases (on the x axis) were knocked down in the female germ line using nanos-Gal4:VP16-driven shRNA. Shown are box plots depicting egg laying rates of these flies (n = 20). Bars indicate the first to third quartiles, horizontal black lines denote the median, and circles denote outliers. Red bars indicate kinases whose reduced expression resulted in decreased numbers of eggs laid, whereas green bars indicate kinases whose reduced expression caused an increase in the number of eggs laid. The blue bar denotes the egg laying rate of flies expressing a control shRNA. Whiskers represent the upper and lower limits of the range. Statistical significance was determined using a Mann-Whitney U test. B, reduced ovarian size upon reduction of CK2 expression. Ovaries from flies of the indicated genotypes were dissected and imaged by light microscopy. Scale bar, 1 mm. C, egg laying rates from flies of the indicated genotypes. Expression of shRNA and/or transgenes was driven using either nanos-Gal4 (green) or matα-Gal4 (blue). Data are presented as in A. D, transgenic expression of epitope-tagged human CK2α and CK2β in the Drosophila female germ line. Ovary lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. Note that CK2α-HA migrates as a doublet.
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    Proteins Co-Immunoprecipitating With NOXO1 in T84 Cells Stimulated by TNFα (5 ng/mL) + IL17 (50 ng/mL) and Related to NADPH Oxidase or Redox Signaling

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

    doi: 10.1016/j.jcmgh.2022.01.003

    Figure Lengend Snippet: Proteins Co-Immunoprecipitating With NOXO1 in T84 Cells Stimulated by TNFα (5 ng/mL) + IL17 (50 ng/mL) and Related to NADPH Oxidase or Redox Signaling

    Article Snippet: Phospho-CK2 Substrate [(pS/pT)DXE] antibodies were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Binding Assay, Protein Binding

    CK2 interacts with NOXO1 in T84 colon epithelial cells under inflammatory conditions. ( A ) Immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon T84 cells stimulated with TNFα (5 ng/mL) or IL17 (50 ng/mL) individually or in combination for 24 hours at 37°C. Representative of 3 independent experiments. ( B ) ROS production was measured by chemiluminescence in T84 cells stimulated as in (A) . n = 3. ( C ) Immunoblots of CK2 α/α′ in NOXO1 IPP from T84 cells stimulated as in (A) . Representative of 4 independent experiments. ( D ) Densitometry analysis of CK2 α/α′ in NOXO1 IPP as in ( C ), normalized to CK2 α/α′expression in cell lysates; n = 4; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05. ( E ) Confocal microcopy of T84 cells co-stimulated or not with TNFα + IL17 for 24 hours at 37°C. NOXO1 ( green ), CK2 α/α′ ( red ), DAPI ( blue ). Scale bar: 10 μm. Representative of 6 independent experiments.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

    doi: 10.1016/j.jcmgh.2022.01.003

    Figure Lengend Snippet: CK2 interacts with NOXO1 in T84 colon epithelial cells under inflammatory conditions. ( A ) Immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon T84 cells stimulated with TNFα (5 ng/mL) or IL17 (50 ng/mL) individually or in combination for 24 hours at 37°C. Representative of 3 independent experiments. ( B ) ROS production was measured by chemiluminescence in T84 cells stimulated as in (A) . n = 3. ( C ) Immunoblots of CK2 α/α′ in NOXO1 IPP from T84 cells stimulated as in (A) . Representative of 4 independent experiments. ( D ) Densitometry analysis of CK2 α/α′ in NOXO1 IPP as in ( C ), normalized to CK2 α/α′expression in cell lysates; n = 4; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05. ( E ) Confocal microcopy of T84 cells co-stimulated or not with TNFα + IL17 for 24 hours at 37°C. NOXO1 ( green ), CK2 α/α′ ( red ), DAPI ( blue ). Scale bar: 10 μm. Representative of 6 independent experiments.

    Article Snippet: Phospho-CK2 Substrate [(pS/pT)DXE] antibodies were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Western Blot, Expressing

    CK2 interacts directly with NOXO1 through the N-terminal region mostly containing the PX domain. ( A ) Coomassie Blue staining of recombinant NOXO1, p47 PHOX , NOXA1, and p67 PHOX . ( B ) Dot-blot analysis of interaction between CK2 and recombinant NOXO1, p47 PHOX , NOXA1, or p67 PHOX . Representative of 3 independent experiments. ( C ) Schematic representation and Coomassie Blue staining of GST fusion proteins of the NOXO1 full-length (β isoform), N-terminal (1-157), and C-terminal regions (232-371). ( D ) Pull-down of CK2 from resting T84 cells lysates by full-length GST-NOXO1, GST-NOXO1 (1-157), GST-NOXO1 (232-371), or GST (control). Representative of 3 independent experiments. ( E ) Densitometry analysis of CK2 α/α′ pull-downed as in ( D ), normalized to GST-fusion proteins; n = 3; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗∗∗ P < 0.0001.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

    doi: 10.1016/j.jcmgh.2022.01.003

    Figure Lengend Snippet: CK2 interacts directly with NOXO1 through the N-terminal region mostly containing the PX domain. ( A ) Coomassie Blue staining of recombinant NOXO1, p47 PHOX , NOXA1, and p67 PHOX . ( B ) Dot-blot analysis of interaction between CK2 and recombinant NOXO1, p47 PHOX , NOXA1, or p67 PHOX . Representative of 3 independent experiments. ( C ) Schematic representation and Coomassie Blue staining of GST fusion proteins of the NOXO1 full-length (β isoform), N-terminal (1-157), and C-terminal regions (232-371). ( D ) Pull-down of CK2 from resting T84 cells lysates by full-length GST-NOXO1, GST-NOXO1 (1-157), GST-NOXO1 (232-371), or GST (control). Representative of 3 independent experiments. ( E ) Densitometry analysis of CK2 α/α′ pull-downed as in ( D ), normalized to GST-fusion proteins; n = 3; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗∗∗ P < 0.0001.

    Article Snippet: Phospho-CK2 Substrate [(pS/pT)DXE] antibodies were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Staining, Recombinant, Dot Blot

    CK2 phosphorylates recombinant NOXO1 in vitro on several sites. ( A ) Autoradiography of recombinant NOXO1 phosphorylated with constitutively active CK2 in the presence of radioactive [γ -32P ] ATP. Time course ( left ), with or without CK2 ( right ). Representative of 3 independent experiments. ( B ) MS/MS protein coverage of recombinant NOXO1 after in vitro phosphorylation by CK2. Peptides were filtered according to 1% false discovery rate that corresponds to an identification score value >37.6 (–10lgP) with the software Peaks Studio Xpro. The confidence of modification sites is estimated by an Ascore, which calculates an ambiguity score as –10 × log 10 (p). The P value indicates the likelihood that the peptide is matched by chance (Ascore = 20 for P value of .01). Only confident modification sites with Ascore >20 were retained and their position labeled ( red ). ( C ) XIC of the heavily C-terminus phosphorylated peptide of m/z 753.997 showing 3 distinct elution peaks for 3 distinct phosphorylation sites. ( D ) Representative MS/MS spectrum of the phosphorylated C-terminus peptide at position 368. The confidence of modification sites is estimated by an Ascore (Ascore = 20 for P value of .01).

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

    doi: 10.1016/j.jcmgh.2022.01.003

    Figure Lengend Snippet: CK2 phosphorylates recombinant NOXO1 in vitro on several sites. ( A ) Autoradiography of recombinant NOXO1 phosphorylated with constitutively active CK2 in the presence of radioactive [γ -32P ] ATP. Time course ( left ), with or without CK2 ( right ). Representative of 3 independent experiments. ( B ) MS/MS protein coverage of recombinant NOXO1 after in vitro phosphorylation by CK2. Peptides were filtered according to 1% false discovery rate that corresponds to an identification score value >37.6 (–10lgP) with the software Peaks Studio Xpro. The confidence of modification sites is estimated by an Ascore, which calculates an ambiguity score as –10 × log 10 (p). The P value indicates the likelihood that the peptide is matched by chance (Ascore = 20 for P value of .01). Only confident modification sites with Ascore >20 were retained and their position labeled ( red ). ( C ) XIC of the heavily C-terminus phosphorylated peptide of m/z 753.997 showing 3 distinct elution peaks for 3 distinct phosphorylation sites. ( D ) Representative MS/MS spectrum of the phosphorylated C-terminus peptide at position 368. The confidence of modification sites is estimated by an Ascore (Ascore = 20 for P value of .01).

    Article Snippet: Phospho-CK2 Substrate [(pS/pT)DXE] antibodies were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Recombinant, In Vitro, Autoradiography, Tandem Mass Spectroscopy, Software, Modification, Labeling

    Inhibition of CK2 enhances ROS production by NOX1 in colon T84 epithelial cells under inflammatory conditions. ( A ) ROS production was measured by chemiluminescence in T84 cells co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 after 24-hour incubation at 37°C. n = 3. ( B ) CK2 activity (top) assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody and NOXO1 expression (middle) in T84 cells co-stimulated as in (A) . Representative of 3 independent experiments. ( C ) Concentration-dependent effect of CX-4549 on ROS production by NOX1 in T84 cells co-stimulated as in (A) in presence or absence of various concentrations of CX-4945 for 24 hours at 37°C. Data were expressed as percentage of control (cells treated with TNFα + IL17 in absence of CX-4549); n = 7 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗ P < .01. ( D ) Concentration-dependent effect of TBBz on ROS production by NOX1 measured by chemiluminescence in T84 cells co-stimulated as in (A) in the presence or absence of various concentrations of TBBz for 24 hours at 37°C. ( E ) Concentration-dependent effect of CX4945 on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM; one-way ANOVA with Dunnett multiple comparisons test; ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. ( F ) Concentration-dependent effect of TBBz on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

    doi: 10.1016/j.jcmgh.2022.01.003

    Figure Lengend Snippet: Inhibition of CK2 enhances ROS production by NOX1 in colon T84 epithelial cells under inflammatory conditions. ( A ) ROS production was measured by chemiluminescence in T84 cells co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 after 24-hour incubation at 37°C. n = 3. ( B ) CK2 activity (top) assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody and NOXO1 expression (middle) in T84 cells co-stimulated as in (A) . Representative of 3 independent experiments. ( C ) Concentration-dependent effect of CX-4549 on ROS production by NOX1 in T84 cells co-stimulated as in (A) in presence or absence of various concentrations of CX-4945 for 24 hours at 37°C. Data were expressed as percentage of control (cells treated with TNFα + IL17 in absence of CX-4549); n = 7 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗∗ P < .01. ( D ) Concentration-dependent effect of TBBz on ROS production by NOX1 measured by chemiluminescence in T84 cells co-stimulated as in (A) in the presence or absence of various concentrations of TBBz for 24 hours at 37°C. ( E ) Concentration-dependent effect of CX4945 on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM; one-way ANOVA with Dunnett multiple comparisons test; ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. ( F ) Concentration-dependent effect of TBBz on proliferation/cytotoxicity of T84 epithelial cells. n = 3 per condition; mean ± SEM.

    Article Snippet: Phospho-CK2 Substrate [(pS/pT)DXE] antibodies were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Inhibition, Incubation, Activity Assay, Expressing, Concentration Assay

    Inhibition of CK2 enhances ROS production by NOX1 in colon organoids under inflammatory conditions. ( A ) Representative immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon organoids stimulated as in <xref ref-type=Figure 1 A for 24 hours at 37°C. ( B ) Representative images of colon organoids established from colon biopsies of control patients. At day 8, organoids were co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 for 24 hours at 37°C. Scale bar: 100 μm. ( C ) ROS production was measured by chemiluminescence in colon organoids treated as in (B). ( D ) Data as in (C) were quantified and expressed as percentage of control (resting cells in the absence of CX-4549); n = 4 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05, ∗∗∗ P < .001. ( E ) ROS production by organoids treated as in ( B) was assessed by NBT reduction. Phase contrast microcopy with ×400 magnification. " width="100%" height="100%">

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

    doi: 10.1016/j.jcmgh.2022.01.003

    Figure Lengend Snippet: Inhibition of CK2 enhances ROS production by NOX1 in colon organoids under inflammatory conditions. ( A ) Representative immunoblots of NOXO1, CK2α/α′, CK2β, and β-actin in colon organoids stimulated as in Figure 1 A for 24 hours at 37°C. ( B ) Representative images of colon organoids established from colon biopsies of control patients. At day 8, organoids were co-stimulated or not with TNFα + IL17 in presence or absence of 1 μmol/L CX-4945 for 24 hours at 37°C. Scale bar: 100 μm. ( C ) ROS production was measured by chemiluminescence in colon organoids treated as in (B). ( D ) Data as in (C) were quantified and expressed as percentage of control (resting cells in the absence of CX-4549); n = 4 per condition; mean ± SEM; one-way ANOVA with Tukey multiple comparisons test; ∗ P < .05, ∗∗∗ P < .001. ( E ) ROS production by organoids treated as in ( B) was assessed by NBT reduction. Phase contrast microcopy with ×400 magnification.

    Article Snippet: Phospho-CK2 Substrate [(pS/pT)DXE] antibodies were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Inhibition, Western Blot

    CK2 activity is highly reduced and NOX1 expression is increased during TNBS-induced acute colitis. ( A ) Left, representative image of colons 24 hours after injection of TNBS or vehicle (CTL); middle, weight/length ratio changes after TNBS treatment; right, macroscopic lesions as assessed by Wallace score. Mean ± SEM, ≥10 animals; Mann-Whitney test; ∗∗∗∗ P < .0001. ( B ) Histologic images of colons 24 hours after TNBS-induced colitis (original magnification, ×200) as compared with CTL (vehicle). Scale bar: 50 μm. ( C ) CK2 activity in cytosolic and membrane fractions of colon homogenates assessed 24 hours after injection of TNBS or vehicle (CTL) with the phospho-CK2-substrate antibody. Four mice are shown (M1 to M4) for each group. ( D ) Densitometry analysis of p-CK2 substrates normalized to β-actin expression. Mean ± SEM from 10 animals; Kruskal-Wallis test with Dunn multiple comparisons test; ∗∗ P < .01, ∗∗∗ P < .001. ( E ) Immunoblots of CK2α/α′ and CK2β in colon homogenates 24 hours after injection of TNBS or vehicle (CTL). Four mice are shown (M1 to M4) for each group. ( F ) Densitometry analysis of CK2α/α′ and CK2β normalized to β-actin expression. Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗∗ P < .01. ( G ) Immunoblots of NOX1 and p22 PHOX in colon homogenates 24 hours after injection of TNBS or vehicle (CTL). Two mice (M1 to M2) in the CTL group and 5 mice (M1 to M5) in the TNBS group are shown. ( H ) Densitometry analysis of NOX1 and p22 PHOX normalized to β-actin expression. Mean ± SEM, ≥8 animals; Mann-Whitney test; ∗∗ P < .01. ( I ) Expression of NOXO1 and NOXA1 RNA messengers during TNBS-induced acute colitis. Relative expression levels for each gene were calculated using the 2 -ΔΔCt method, with normalization to the average and GAPDH housekeeping genes. Mean ± SEM, 9 animals; Mann-Whitney test; ∗∗ P < .01.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

    doi: 10.1016/j.jcmgh.2022.01.003

    Figure Lengend Snippet: CK2 activity is highly reduced and NOX1 expression is increased during TNBS-induced acute colitis. ( A ) Left, representative image of colons 24 hours after injection of TNBS or vehicle (CTL); middle, weight/length ratio changes after TNBS treatment; right, macroscopic lesions as assessed by Wallace score. Mean ± SEM, ≥10 animals; Mann-Whitney test; ∗∗∗∗ P < .0001. ( B ) Histologic images of colons 24 hours after TNBS-induced colitis (original magnification, ×200) as compared with CTL (vehicle). Scale bar: 50 μm. ( C ) CK2 activity in cytosolic and membrane fractions of colon homogenates assessed 24 hours after injection of TNBS or vehicle (CTL) with the phospho-CK2-substrate antibody. Four mice are shown (M1 to M4) for each group. ( D ) Densitometry analysis of p-CK2 substrates normalized to β-actin expression. Mean ± SEM from 10 animals; Kruskal-Wallis test with Dunn multiple comparisons test; ∗∗ P < .01, ∗∗∗ P < .001. ( E ) Immunoblots of CK2α/α′ and CK2β in colon homogenates 24 hours after injection of TNBS or vehicle (CTL). Four mice are shown (M1 to M4) for each group. ( F ) Densitometry analysis of CK2α/α′ and CK2β normalized to β-actin expression. Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗∗ P < .01. ( G ) Immunoblots of NOX1 and p22 PHOX in colon homogenates 24 hours after injection of TNBS or vehicle (CTL). Two mice (M1 to M2) in the CTL group and 5 mice (M1 to M5) in the TNBS group are shown. ( H ) Densitometry analysis of NOX1 and p22 PHOX normalized to β-actin expression. Mean ± SEM, ≥8 animals; Mann-Whitney test; ∗∗ P < .01. ( I ) Expression of NOXO1 and NOXA1 RNA messengers during TNBS-induced acute colitis. Relative expression levels for each gene were calculated using the 2 -ΔΔCt method, with normalization to the average and GAPDH housekeeping genes. Mean ± SEM, 9 animals; Mann-Whitney test; ∗∗ P < .01.

    Article Snippet: Phospho-CK2 Substrate [(pS/pT)DXE] antibodies were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Activity Assay, Expressing, Injection, MANN-WHITNEY, Western Blot

    Effect of CK2β and CK2α overexpression on CK2 activity and ROS production in colon T84 epithelial cells. ( A ) Immunoblots of HA-CK2β and Myc-CK2α overexpressed individually in colon T84 cells. ( B ) CK2 activity assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody in T84 cells overexpressing CK2β or CK2α. ( C ) ROS production was measured by chemiluminescence in T84 cells overexpressing CK2β or CK2α. n = 3. ( D ) Effect of increasing concentrations of recombinant CK2β subunit on phosphorylation of NOXO1 by CK2 in vitro . Autoradiography (Autorad.) and Ponceau-Red are shown.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

    doi: 10.1016/j.jcmgh.2022.01.003

    Figure Lengend Snippet: Effect of CK2β and CK2α overexpression on CK2 activity and ROS production in colon T84 epithelial cells. ( A ) Immunoblots of HA-CK2β and Myc-CK2α overexpressed individually in colon T84 cells. ( B ) CK2 activity assessed with the phospho-CK2-substrate [(pS/pT)DXE] antibody in T84 cells overexpressing CK2β or CK2α. ( C ) ROS production was measured by chemiluminescence in T84 cells overexpressing CK2β or CK2α. n = 3. ( D ) Effect of increasing concentrations of recombinant CK2β subunit on phosphorylation of NOXO1 by CK2 in vitro . Autoradiography (Autorad.) and Ponceau-Red are shown.

    Article Snippet: Phospho-CK2 Substrate [(pS/pT)DXE] antibodies were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Over Expression, Activity Assay, Western Blot, Recombinant, In Vitro, Autoradiography

    The highly selective CK2 inhibitor CX-4945 exacerbates TNBS-induced colitis. ( A ) Representative images of colons 24 hours after injection of TNBS or vehicle (CTL) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. ( B ) Histologic images of colons 24 hours after TNBS-induced colitis (original magnification, ×200) as compared with CTL (vehicle) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. Scale bar: 50 μm. ( C ) Histologic damage of colon as assessed by the Ameho score in mice treated as in (B) . Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗ P < .05. ( D ) Secretion of CXCL1 (pg. mg -1 protein) in colons of mice treated as in (B). Mean ± SEM from 4 animals (CTL groups with and without CX-4945) to 6 animals (TNBS groups with and without CX-4945), Mann-Whitney test; ∗ P < .05. ( E ) ROS production assessed ex vivo by NBT reduction in colon tissue of mice treated as in (B) . Formazan deposits were examined macroscopically. Representative of 4 mice in the CTL groups with and without CX-4945 and at least 7 mice in the TNBS groups with and without CX-4945. ( F ) MDA (nmol. mg -1 protein) content in the colon of mice treated as in (B) , normalized as compared with control. Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗ P < .05.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

    doi: 10.1016/j.jcmgh.2022.01.003

    Figure Lengend Snippet: The highly selective CK2 inhibitor CX-4945 exacerbates TNBS-induced colitis. ( A ) Representative images of colons 24 hours after injection of TNBS or vehicle (CTL) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. ( B ) Histologic images of colons 24 hours after TNBS-induced colitis (original magnification, ×200) as compared with CTL (vehicle) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. Scale bar: 50 μm. ( C ) Histologic damage of colon as assessed by the Ameho score in mice treated as in (B) . Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗ P < .05. ( D ) Secretion of CXCL1 (pg. mg -1 protein) in colons of mice treated as in (B). Mean ± SEM from 4 animals (CTL groups with and without CX-4945) to 6 animals (TNBS groups with and without CX-4945), Mann-Whitney test; ∗ P < .05. ( E ) ROS production assessed ex vivo by NBT reduction in colon tissue of mice treated as in (B) . Formazan deposits were examined macroscopically. Representative of 4 mice in the CTL groups with and without CX-4945 and at least 7 mice in the TNBS groups with and without CX-4945. ( F ) MDA (nmol. mg -1 protein) content in the colon of mice treated as in (B) , normalized as compared with control. Mean ± SEM, ≥6 animals; Mann-Whitney test; ∗ P < .05.

    Article Snippet: Phospho-CK2 Substrate [(pS/pT)DXE] antibodies were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Injection, MANN-WHITNEY, Ex Vivo

    Expression of NOX1 and p22 PHOX during TNBS-induced acute colitis in the presence or absence of CX-4945. Immunoblots of NOX1 and p22 PHOX in colon homogenates 24 hours after injection of TNBS or vehicle (CTL) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. Results from 2 mice (M1 to M2) in the CTL group and 4 mice (M1 to M4) in the TNBS group are shown.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation

    doi: 10.1016/j.jcmgh.2022.01.003

    Figure Lengend Snippet: Expression of NOX1 and p22 PHOX during TNBS-induced acute colitis in the presence or absence of CX-4945. Immunoblots of NOX1 and p22 PHOX in colon homogenates 24 hours after injection of TNBS or vehicle (CTL) in mice treated with the CK2 inhibitor CX-4945 (25 mg/kg) or DMSO. Results from 2 mice (M1 to M2) in the CTL group and 4 mice (M1 to M4) in the TNBS group are shown.

    Article Snippet: Phospho-CK2 Substrate [(pS/pT)DXE] antibodies were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Expressing, Western Blot, Injection

    List of commercial primary antibodies used for Western blot.

    Journal: Frontiers in Neuroscience

    Article Title: GPR68 Contributes to Persistent Acidosis-Induced Activation of AGC Kinases and Tyrosine Phosphorylation in Organotypic Hippocampal Slices

    doi: 10.3389/fnins.2021.692217

    Figure Lengend Snippet: List of commercial primary antibodies used for Western blot.

    Article Snippet: Phospho-(Ser/Thr) CK2 Substrate (rabbit) , (S*/T*)DXE , 1:2,000 , Cell Signaling Technology , 8738S.

    Techniques: Western Blot

    List of commercial primary antibodies used for Western blot.

    Journal: Frontiers in Neuroscience

    Article Title: GPR68 Contributes to Persistent Acidosis-Induced Activation of AGC Kinases and Tyrosine Phosphorylation in Organotypic Hippocampal Slices

    doi: 10.3389/fnins.2021.692217

    Figure Lengend Snippet: List of commercial primary antibodies used for Western blot.

    Article Snippet: Phospho-(Ser/Thr) CK2 Substrate (rabbit) , (S*/T*)DXE , 1:2,000 , Cell Signaling Technology , 8738S.

    Techniques: Western Blot

    CK2 inhibition exerts disease modifying properties. (A) Time frame of the experiment. TBB was administered four times in vivo on days “−3” (i.e., 3 days before status epilepticus) through “0” (i.e., day of pilocarpine-induced status epilepticus). All experiments thereafter were performed during the chronic stage of recurrent seizures. (B–D) Latency to the first stage 4 or 5 seizure after pilocarpine injection in vehicle-pretreated (B) and TBB-pretreated (C) rats. The median latency was significantly (*** P < 0.001, Mann-Whitney U-test) prolonged in the TBB-pretreated group (D) . (E) Number of interictal spikes per hour (left y -axis) in the electroencephalogram (EEG) during 3 days (97–100 post-SE). Note that the number of daily seizures during this period is also depicted (diamonds, right y -axis). There was a significant group effect of TBB pretreatment (** P < 0.01, two-way ANOVA with Tukey post hoc test). (F) Number of generalized seizures (at least stage 3) at daylight (from 06:00 to 18:00 h) for both animal groups as grand average for the entire recording period. (G) Seizure burden progression as fold change of seizure rates during epileptogenesis. The variability was significantly reduced in TBB-pretreated epileptic animals (** P < 0.01, F -test). (H) Relative risk reduction (RRR) of seizure progression by TBB pretreatment was significant (* P < 0.05, t -test).

    Journal: Frontiers in Cellular Neuroscience

    Article Title: CK2 Inhibition Prior to Status Epilepticus Persistently Enhances K Ca 2 Function in CA1 Which Slows Down Disease Progression

    doi: 10.3389/fncel.2020.00033

    Figure Lengend Snippet: CK2 inhibition exerts disease modifying properties. (A) Time frame of the experiment. TBB was administered four times in vivo on days “−3” (i.e., 3 days before status epilepticus) through “0” (i.e., day of pilocarpine-induced status epilepticus). All experiments thereafter were performed during the chronic stage of recurrent seizures. (B–D) Latency to the first stage 4 or 5 seizure after pilocarpine injection in vehicle-pretreated (B) and TBB-pretreated (C) rats. The median latency was significantly (*** P < 0.001, Mann-Whitney U-test) prolonged in the TBB-pretreated group (D) . (E) Number of interictal spikes per hour (left y -axis) in the electroencephalogram (EEG) during 3 days (97–100 post-SE). Note that the number of daily seizures during this period is also depicted (diamonds, right y -axis). There was a significant group effect of TBB pretreatment (** P < 0.01, two-way ANOVA with Tukey post hoc test). (F) Number of generalized seizures (at least stage 3) at daylight (from 06:00 to 18:00 h) for both animal groups as grand average for the entire recording period. (G) Seizure burden progression as fold change of seizure rates during epileptogenesis. The variability was significantly reduced in TBB-pretreated epileptic animals (** P < 0.01, F -test). (H) Relative risk reduction (RRR) of seizure progression by TBB pretreatment was significant (* P < 0.05, t -test).

    Article Snippet: Then, according to CK2 immunoreaction, the rabbit monoclonal anti-GAPDH primary antibody (Cell Signaling; 1:5000) and a secondary antibody (goat anti-rabbit IRDye 800CW, Odyssey; 1:5000) was loaded.

    Techniques: Inhibition, In Vivo, Injection, MANN-WHITNEY

    CK2 inhibition facilitates CA1 plasticity. (A) Sample traces of CA1 stratum radiatum field excitatory postsynaptic potentials (fEPSP) following Schaffer collateral stimulation in control (A 1 ) and epileptic animals (A 2 ). (B) Input–output curves of all four groups without significant group differences. (C) Paired-pulse ratios (PPRs) were similar among all four experimental groups. Short-term plasticity during five stimuli at frequencies of 10, 25, and 33 Hz (D) , and subsequent changes of fEPSP slope (E) and PPR (F) after this short-term plasticity paradigm. Only slices from the TBB-pretreated epileptic group showed significantly facilitated plasticity. * P < 0.05.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: CK2 Inhibition Prior to Status Epilepticus Persistently Enhances K Ca 2 Function in CA1 Which Slows Down Disease Progression

    doi: 10.3389/fncel.2020.00033

    Figure Lengend Snippet: CK2 inhibition facilitates CA1 plasticity. (A) Sample traces of CA1 stratum radiatum field excitatory postsynaptic potentials (fEPSP) following Schaffer collateral stimulation in control (A 1 ) and epileptic animals (A 2 ). (B) Input–output curves of all four groups without significant group differences. (C) Paired-pulse ratios (PPRs) were similar among all four experimental groups. Short-term plasticity during five stimuli at frequencies of 10, 25, and 33 Hz (D) , and subsequent changes of fEPSP slope (E) and PPR (F) after this short-term plasticity paradigm. Only slices from the TBB-pretreated epileptic group showed significantly facilitated plasticity. * P < 0.05.

    Article Snippet: Then, according to CK2 immunoreaction, the rabbit monoclonal anti-GAPDH primary antibody (Cell Signaling; 1:5000) and a secondary antibody (goat anti-rabbit IRDye 800CW, Odyssey; 1:5000) was loaded.

    Techniques: Inhibition

    CK2 inhibition reduces bursting in CA1. (A) Sample traces of CA1 pyramidal cell intracellular recordings from a vehicle-pretreated epileptic rat (Pilo, black traces) and a TBB-pretreated epileptic rat (Pilo-TBB, blue traces). Note the bursting of the upper traces upon just suprathreshold stimulation. (B) Proportions of burster and non-burster neurons in the four experimental groups (* P < 0.05). (C,D) Afterdepolarizing potential (ADP) with different pulse durations ( d = 3, 5, 7 ms). The area under curve of the ADP (starting from d + 5 ms, indicated in gray in C ) of cells from vehicle-pretreated epileptic animals (Pilo) was significant against the ADP of all other experimental groups (* P < 0.05, three-way ANOVA with Tukey post hoc test).

    Journal: Frontiers in Cellular Neuroscience

    Article Title: CK2 Inhibition Prior to Status Epilepticus Persistently Enhances K Ca 2 Function in CA1 Which Slows Down Disease Progression

    doi: 10.3389/fncel.2020.00033

    Figure Lengend Snippet: CK2 inhibition reduces bursting in CA1. (A) Sample traces of CA1 pyramidal cell intracellular recordings from a vehicle-pretreated epileptic rat (Pilo, black traces) and a TBB-pretreated epileptic rat (Pilo-TBB, blue traces). Note the bursting of the upper traces upon just suprathreshold stimulation. (B) Proportions of burster and non-burster neurons in the four experimental groups (* P < 0.05). (C,D) Afterdepolarizing potential (ADP) with different pulse durations ( d = 3, 5, 7 ms). The area under curve of the ADP (starting from d + 5 ms, indicated in gray in C ) of cells from vehicle-pretreated epileptic animals (Pilo) was significant against the ADP of all other experimental groups (* P < 0.05, three-way ANOVA with Tukey post hoc test).

    Article Snippet: Then, according to CK2 immunoreaction, the rabbit monoclonal anti-GAPDH primary antibody (Cell Signaling; 1:5000) and a secondary antibody (goat anti-rabbit IRDye 800CW, Odyssey; 1:5000) was loaded.

    Techniques: Inhibition

    CK2 inhibition modifies KC2 function. (A) Bar graph of the mAHP-mediating current in all four experimental groups. CA1 cells from vehicle-pretreated epileptic animals (Pilo) showed significantly less mAHP currents than control cells and cells from TBB-pretreated epileptic animals. (B) The sAHP charge transfer is significantly smaller in epileptic animals than in controls. TBB has no effect on the sAHP charge transfer. (C) Quantitative RT-PCR analysis of the CA1 subfield reveals that tissue from TBB-pretreated control animals contained significantly more K Ca 2.2 mRNA than vehicle-pretreated controls and TBB-pretreated epileptic rats. (D) Western blot analysis of CK2 protein in TBB-pretreated tissue. Note the region-specific differences between control and epileptic tissues. * P < 0.05.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: CK2 Inhibition Prior to Status Epilepticus Persistently Enhances K Ca 2 Function in CA1 Which Slows Down Disease Progression

    doi: 10.3389/fncel.2020.00033

    Figure Lengend Snippet: CK2 inhibition modifies KC2 function. (A) Bar graph of the mAHP-mediating current in all four experimental groups. CA1 cells from vehicle-pretreated epileptic animals (Pilo) showed significantly less mAHP currents than control cells and cells from TBB-pretreated epileptic animals. (B) The sAHP charge transfer is significantly smaller in epileptic animals than in controls. TBB has no effect on the sAHP charge transfer. (C) Quantitative RT-PCR analysis of the CA1 subfield reveals that tissue from TBB-pretreated control animals contained significantly more K Ca 2.2 mRNA than vehicle-pretreated controls and TBB-pretreated epileptic rats. (D) Western blot analysis of CK2 protein in TBB-pretreated tissue. Note the region-specific differences between control and epileptic tissues. * P < 0.05.

    Article Snippet: Then, according to CK2 immunoreaction, the rabbit monoclonal anti-GAPDH primary antibody (Cell Signaling; 1:5000) and a secondary antibody (goat anti-rabbit IRDye 800CW, Odyssey; 1:5000) was loaded.

    Techniques: Inhibition, Quantitative RT-PCR, Western Blot

    CK2 inhibition modifies HCN channel function. (A) Sample traces of the membrane potential following hyperpolarizing and depolarizing pulse steps in intracellular recordings from CA1 neurons (gray: control, black: vehicle-pretreated epileptic, blue: TBB-pretreated epileptic). (B) Bar graphs of the train-AHP amplitude and voltage sag obtained from recordings shown in A . The train-AHP was significantly smaller in epileptic tissues regardless of the treatment with TBB. In contrast, the voltage sag was enhanced in TBB-pretreated tissue from both control and epileptic animals. Quantitative RT-PCR analysis of the CA1 subfield of all four HCN channel isoforms revealed significant and inverse expression changes for HCN1 (C) and HCN3 (E) , but no changes for HCN2 (D) and HCN4 (F) . * P < 0.05.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: CK2 Inhibition Prior to Status Epilepticus Persistently Enhances K Ca 2 Function in CA1 Which Slows Down Disease Progression

    doi: 10.3389/fncel.2020.00033

    Figure Lengend Snippet: CK2 inhibition modifies HCN channel function. (A) Sample traces of the membrane potential following hyperpolarizing and depolarizing pulse steps in intracellular recordings from CA1 neurons (gray: control, black: vehicle-pretreated epileptic, blue: TBB-pretreated epileptic). (B) Bar graphs of the train-AHP amplitude and voltage sag obtained from recordings shown in A . The train-AHP was significantly smaller in epileptic tissues regardless of the treatment with TBB. In contrast, the voltage sag was enhanced in TBB-pretreated tissue from both control and epileptic animals. Quantitative RT-PCR analysis of the CA1 subfield of all four HCN channel isoforms revealed significant and inverse expression changes for HCN1 (C) and HCN3 (E) , but no changes for HCN2 (D) and HCN4 (F) . * P < 0.05.

    Article Snippet: Then, according to CK2 immunoreaction, the rabbit monoclonal anti-GAPDH primary antibody (Cell Signaling; 1:5000) and a secondary antibody (goat anti-rabbit IRDye 800CW, Odyssey; 1:5000) was loaded.

    Techniques: Inhibition, Quantitative RT-PCR, Expressing

    CK2 inhibition enhances train-AHP and reduces excitability in HCN-blocking conditions. (A) Proportions of burster and non-burster neurons in the four experimental groups in the presence of the HCN channel blocker ZD7288. (B) Sample traces of intracellular recordings following prolonged depolarization (600 ms) used for train-AHP amplitude (e.g., see blue arrow) and number of action potentials. For comparison, sample traces in the absence of ZD7288 were also given (left side). (C) Bar graph of the train-AHP amplitude shows a significantly increased train-AHP in cells from TBB-pretreated epileptic rats (blue bar graph). For the sake of clarity, the train-AHP in the absence of ZD7288 (gray bar graphs, values from ) are also shown. Significant effects were detected between animal groups (Ctrl versus Pilo, * P < 0.05) and pretreatment groups (vehicle versus TBB, * P < 0.05), but not for the ZD7288 effect by three-way ANOVA with Holm–Sidak post hoc tests. However, there was a significant interaction between ZD7288 and pretreatment with TBB or vehicle (* P < 0.05). (D) Number of action potentials during 600 ms depolarization (from 100 to 500 pA). Two-way ANOVA with Tukey post hoc tests showed a trend effect between animal groups (Ctrl versus Pilo, + P = 0.08) and a significant effect treatment groups (vehicle versus TBB, * P < 0.05).

    Journal: Frontiers in Cellular Neuroscience

    Article Title: CK2 Inhibition Prior to Status Epilepticus Persistently Enhances K Ca 2 Function in CA1 Which Slows Down Disease Progression

    doi: 10.3389/fncel.2020.00033

    Figure Lengend Snippet: CK2 inhibition enhances train-AHP and reduces excitability in HCN-blocking conditions. (A) Proportions of burster and non-burster neurons in the four experimental groups in the presence of the HCN channel blocker ZD7288. (B) Sample traces of intracellular recordings following prolonged depolarization (600 ms) used for train-AHP amplitude (e.g., see blue arrow) and number of action potentials. For comparison, sample traces in the absence of ZD7288 were also given (left side). (C) Bar graph of the train-AHP amplitude shows a significantly increased train-AHP in cells from TBB-pretreated epileptic rats (blue bar graph). For the sake of clarity, the train-AHP in the absence of ZD7288 (gray bar graphs, values from ) are also shown. Significant effects were detected between animal groups (Ctrl versus Pilo, * P < 0.05) and pretreatment groups (vehicle versus TBB, * P < 0.05), but not for the ZD7288 effect by three-way ANOVA with Holm–Sidak post hoc tests. However, there was a significant interaction between ZD7288 and pretreatment with TBB or vehicle (* P < 0.05). (D) Number of action potentials during 600 ms depolarization (from 100 to 500 pA). Two-way ANOVA with Tukey post hoc tests showed a trend effect between animal groups (Ctrl versus Pilo, + P = 0.08) and a significant effect treatment groups (vehicle versus TBB, * P < 0.05).

    Article Snippet: Then, according to CK2 immunoreaction, the rabbit monoclonal anti-GAPDH primary antibody (Cell Signaling; 1:5000) and a secondary antibody (goat anti-rabbit IRDye 800CW, Odyssey; 1:5000) was loaded.

    Techniques: Inhibition, Blocking Assay

    CK2 kinase activity is essential for Drosophila oogenesis. A, identification of novel kinase regulators of Drosophila oogenesis. The indicated 78 kinases (on the x axis) were knocked down in the female germ line using nanos-Gal4:VP16-driven shRNA. Shown are box plots depicting egg laying rates of these flies (n = 20). Bars indicate the first to third quartiles, horizontal black lines denote the median, and circles denote outliers. Red bars indicate kinases whose reduced expression resulted in decreased numbers of eggs laid, whereas green bars indicate kinases whose reduced expression caused an increase in the number of eggs laid. The blue bar denotes the egg laying rate of flies expressing a control shRNA. Whiskers represent the upper and lower limits of the range. Statistical significance was determined using a Mann-Whitney U test. B, reduced ovarian size upon reduction of CK2 expression. Ovaries from flies of the indicated genotypes were dissected and imaged by light microscopy. Scale bar, 1 mm. C, egg laying rates from flies of the indicated genotypes. Expression of shRNA and/or transgenes was driven using either nanos-Gal4 (green) or matα-Gal4 (blue). Data are presented as in A. D, transgenic expression of epitope-tagged human CK2α and CK2β in the Drosophila female germ line. Ovary lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. Note that CK2α-HA migrates as a doublet.

    Journal: The Journal of Biological Chemistry

    Article Title: The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis

    doi: 10.1074/jbc.M117.814657

    Figure Lengend Snippet: CK2 kinase activity is essential for Drosophila oogenesis. A, identification of novel kinase regulators of Drosophila oogenesis. The indicated 78 kinases (on the x axis) were knocked down in the female germ line using nanos-Gal4:VP16-driven shRNA. Shown are box plots depicting egg laying rates of these flies (n = 20). Bars indicate the first to third quartiles, horizontal black lines denote the median, and circles denote outliers. Red bars indicate kinases whose reduced expression resulted in decreased numbers of eggs laid, whereas green bars indicate kinases whose reduced expression caused an increase in the number of eggs laid. The blue bar denotes the egg laying rate of flies expressing a control shRNA. Whiskers represent the upper and lower limits of the range. Statistical significance was determined using a Mann-Whitney U test. B, reduced ovarian size upon reduction of CK2 expression. Ovaries from flies of the indicated genotypes were dissected and imaged by light microscopy. Scale bar, 1 mm. C, egg laying rates from flies of the indicated genotypes. Expression of shRNA and/or transgenes was driven using either nanos-Gal4 (green) or matα-Gal4 (blue). Data are presented as in A. D, transgenic expression of epitope-tagged human CK2α and CK2β in the Drosophila female germ line. Ovary lysates were prepared, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. Note that CK2α-HA migrates as a doublet.

    Article Snippet: Anti-HA and anti-phospho-CK2 substrate ((pS/pT)D X E where pS is phosphoserine and pT is phosphothreonine) antibodies were from Cell Signaling Technology.

    Techniques: Activity Assay, shRNA, Expressing, MANN-WHITNEY, Light Microscopy, Transgenic Assay, SDS Page

    A biochemical screen identifies substrates of CK2 in the Drosophila ovary. A, schematic of the strategy used to identify novel ovarian substrates of CK2. B, experimental validation of the approach outlined in A. Ovary lysate was incubated for 30 min at 30 °C with or without recombinant human CK2 holoenzyme and in the presence or absence of CX-4945 (100 μm) as indicated. GTPγS (2.5 mm) was then added, and the reactions were incubated further for 1 min at 30 °C followed by treatment with PNBM for 60 min at room temperature. Kinase reactions were subsequently resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, immunoprecipitation (IP) of putative CK2 substrates from Drosophila ovary lysate. An anti-thiophosphate ester antibody was used to immunoprecipitate thiophosphorylated proteins. Immunoprecipitated proteins were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. The yellow asterisk indicates the position of the band that was excised from a corresponding Coomassie Blue–stained gel for protein identification by LC-MS/MS. D, primary amino acid sequence of Jabba (isoform F). Highlighted in red are unique peptides, representing 7% coverage of the protein, identified by mass spectrometry.

    Journal: The Journal of Biological Chemistry

    Article Title: The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis

    doi: 10.1074/jbc.M117.814657

    Figure Lengend Snippet: A biochemical screen identifies substrates of CK2 in the Drosophila ovary. A, schematic of the strategy used to identify novel ovarian substrates of CK2. B, experimental validation of the approach outlined in A. Ovary lysate was incubated for 30 min at 30 °C with or without recombinant human CK2 holoenzyme and in the presence or absence of CX-4945 (100 μm) as indicated. GTPγS (2.5 mm) was then added, and the reactions were incubated further for 1 min at 30 °C followed by treatment with PNBM for 60 min at room temperature. Kinase reactions were subsequently resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, immunoprecipitation (IP) of putative CK2 substrates from Drosophila ovary lysate. An anti-thiophosphate ester antibody was used to immunoprecipitate thiophosphorylated proteins. Immunoprecipitated proteins were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. The yellow asterisk indicates the position of the band that was excised from a corresponding Coomassie Blue–stained gel for protein identification by LC-MS/MS. D, primary amino acid sequence of Jabba (isoform F). Highlighted in red are unique peptides, representing 7% coverage of the protein, identified by mass spectrometry.

    Article Snippet: Anti-HA and anti-phospho-CK2 substrate ((pS/pT)D X E where pS is phosphoserine and pT is phosphothreonine) antibodies were from Cell Signaling Technology.

    Techniques: Incubation, Recombinant, SDS Page, Immunoprecipitation, Western Blot, Staining, Liquid Chromatography with Mass Spectroscopy, Sequencing, Mass Spectrometry

    Jabba is a novel CK2 substrate. A, in vitro kinase assay using GST-Jabba and CK2. GST-Jabba (or no substrate) was incubated in the presence or absence of recombinant CK2 holoenzyme and ATPγS followed by treatment with PNBM. Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. B, inhibition of CK2 kinase activity using the small-molecule inhibitor CX-4945. HEK293 cells were treated (or not) with the indicated concentrations of CX-4945 for 24 h. Cell lysates (40 μg of total protein) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, HEK293 cells were transfected (or not) with a plasmid encoding GFP-Jabba. Cell lysates (5 μg of total protein) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. D, HEK293 cells were cotransfected with plasmids encoding GFP-Jabba and pCMV-HA (empty vector) or pCMV-CK2α-HA. Cells were lysed followed by immunoprecipitation of GFP-Jabba and immunoblotting of the precipitated protein with the indicated antibodies. Western blots shown are representative of three independent experiments. IP, immunoprecipitate; WCL, whole cell lysate.

    Journal: The Journal of Biological Chemistry

    Article Title: The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis

    doi: 10.1074/jbc.M117.814657

    Figure Lengend Snippet: Jabba is a novel CK2 substrate. A, in vitro kinase assay using GST-Jabba and CK2. GST-Jabba (or no substrate) was incubated in the presence or absence of recombinant CK2 holoenzyme and ATPγS followed by treatment with PNBM. Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. B, inhibition of CK2 kinase activity using the small-molecule inhibitor CX-4945. HEK293 cells were treated (or not) with the indicated concentrations of CX-4945 for 24 h. Cell lysates (40 μg of total protein) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. C, HEK293 cells were transfected (or not) with a plasmid encoding GFP-Jabba. Cell lysates (5 μg of total protein) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. D, HEK293 cells were cotransfected with plasmids encoding GFP-Jabba and pCMV-HA (empty vector) or pCMV-CK2α-HA. Cells were lysed followed by immunoprecipitation of GFP-Jabba and immunoblotting of the precipitated protein with the indicated antibodies. Western blots shown are representative of three independent experiments. IP, immunoprecipitate; WCL, whole cell lysate.

    Article Snippet: Anti-HA and anti-phospho-CK2 substrate ((pS/pT)D X E where pS is phosphoserine and pT is phosphothreonine) antibodies were from Cell Signaling Technology.

    Techniques: In Vitro, Kinase Assay, Incubation, Recombinant, SDS Page, Western Blot, Inhibition, Activity Assay, Transfection, Plasmid Preparation, Immunoprecipitation

    CK2 phosphorylates multiple regions of Jabba. A, schematic diagram of Jabba (isoform F) depicting various truncation fragments generated. B, in vitro kinase assay using GST-Jabba(222–273) and CK2. GST or GST-Jabba(222–273) was incubated with recombinant CK2 holoenzyme and ATPγS in the presence or absence of CX-4945 (10 μm) followed by treatment with PNBM. Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. C, in vitro kinase assay using GST-Jabba(274–537) and CK2. GST or GST-Jabba(274–537) was incubated with recombinant CK2 holoenzyme and ATP in the presence or absence of CX-4945 (10 μm). Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis

    doi: 10.1074/jbc.M117.814657

    Figure Lengend Snippet: CK2 phosphorylates multiple regions of Jabba. A, schematic diagram of Jabba (isoform F) depicting various truncation fragments generated. B, in vitro kinase assay using GST-Jabba(222–273) and CK2. GST or GST-Jabba(222–273) was incubated with recombinant CK2 holoenzyme and ATPγS in the presence or absence of CX-4945 (10 μm) followed by treatment with PNBM. Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments. C, in vitro kinase assay using GST-Jabba(274–537) and CK2. GST or GST-Jabba(274–537) was incubated with recombinant CK2 holoenzyme and ATP in the presence or absence of CX-4945 (10 μm). Kinase reactions were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. Western blots shown are representative of three independent experiments.

    Article Snippet: Anti-HA and anti-phospho-CK2 substrate ((pS/pT)D X E where pS is phosphoserine and pT is phosphothreonine) antibodies were from Cell Signaling Technology.

    Techniques: Generated, In Vitro, Kinase Assay, Incubation, Recombinant, SDS Page, Western Blot

    Jabba localizes to lipid droplets in a CK2-independent manner. A, 3T3-L1 preadipocytes were cotransfected with plasmids encoding myc-perilipin-3 (Plin-3) and GFP. Cells were treated with oleic acid. Cells were then treated with DMSO (top panels) or CX-4945 (10 μm) (bottom panels), fixed, and processed for immunofluorescence microscopy with anti-myc antibodies. Cells were counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm. B, 3T3-L1 preadipocytes were cotransfected with plasmids encoding myc-perlipin-3 (Plin-3) and GFP-Jabba and incubated with oleic acid. Cells were then treated with DMSO (top panels) or CX-4945 (10 μm) (bottom panels), fixed, and processed for immunofluorescence microscopy with anti-myc antibody. Cells were counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis

    doi: 10.1074/jbc.M117.814657

    Figure Lengend Snippet: Jabba localizes to lipid droplets in a CK2-independent manner. A, 3T3-L1 preadipocytes were cotransfected with plasmids encoding myc-perilipin-3 (Plin-3) and GFP. Cells were treated with oleic acid. Cells were then treated with DMSO (top panels) or CX-4945 (10 μm) (bottom panels), fixed, and processed for immunofluorescence microscopy with anti-myc antibodies. Cells were counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm. B, 3T3-L1 preadipocytes were cotransfected with plasmids encoding myc-perlipin-3 (Plin-3) and GFP-Jabba and incubated with oleic acid. Cells were then treated with DMSO (top panels) or CX-4945 (10 μm) (bottom panels), fixed, and processed for immunofluorescence microscopy with anti-myc antibody. Cells were counterstained with DAPI to label nuclei (blue). Scale bars, 10 μm.

    Article Snippet: Anti-HA and anti-phospho-CK2 substrate ((pS/pT)D X E where pS is phosphoserine and pT is phosphothreonine) antibodies were from Cell Signaling Technology.

    Techniques: Immunofluorescence, Microscopy, Incubation

    Jabba and CK2 regulate lipid metabolism during Drosophila oogenesis. A, ovaries from flies of the indicated genotypes were dissected and stained with Oil Red O for visualization of neutral lipids. Shown are representative stage 10 egg chambers. Scale bar, 100 μm. B, quantitation of total triglyceride levels from ovaries of flies of the indicated genotypes. Data are presented as the mean (n = 4) with error bars denoting S.D. * indicates a p value <0.05. ns, not significant. C, confocal immunofluorescence microscopy of egg chambers from flies of the indicated genotypes. Dissected egg chambers were fixed and stained with anti-myc antibody to detect Jabba-labeled lipid droplets (green) and counterstained with rhodamine phalloidin to label F-actin (red). Shown are single optical confocal sections. Scale bars, 50 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: The protein kinase CK2 substrate Jabba modulates lipid metabolism during Drosophila oogenesis

    doi: 10.1074/jbc.M117.814657

    Figure Lengend Snippet: Jabba and CK2 regulate lipid metabolism during Drosophila oogenesis. A, ovaries from flies of the indicated genotypes were dissected and stained with Oil Red O for visualization of neutral lipids. Shown are representative stage 10 egg chambers. Scale bar, 100 μm. B, quantitation of total triglyceride levels from ovaries of flies of the indicated genotypes. Data are presented as the mean (n = 4) with error bars denoting S.D. * indicates a p value <0.05. ns, not significant. C, confocal immunofluorescence microscopy of egg chambers from flies of the indicated genotypes. Dissected egg chambers were fixed and stained with anti-myc antibody to detect Jabba-labeled lipid droplets (green) and counterstained with rhodamine phalloidin to label F-actin (red). Shown are single optical confocal sections. Scale bars, 50 μm.

    Article Snippet: Anti-HA and anti-phospho-CK2 substrate ((pS/pT)D X E where pS is phosphoserine and pT is phosphothreonine) antibodies were from Cell Signaling Technology.

    Techniques: Staining, Quantitation Assay, Immunofluorescence, Microscopy, Labeling