recombinant human ck2β protein  (GeneTex)

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: In some assays recombinant human CK2β protein (GeneTex Inc, Irvine, CA) was added.

Techniques: Western Blot, Expressing

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: In some assays recombinant human CK2β protein (GeneTex Inc, Irvine, CA) was added.

Techniques: Staining, Recombinant, Dot Blot, Control

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: In some assays recombinant human CK2β protein (GeneTex Inc, Irvine, CA) was added.

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

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: In some assays recombinant human CK2β protein (GeneTex Inc, Irvine, CA) was added.

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

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: In some assays recombinant human CK2β protein (GeneTex Inc, Irvine, CA) was added.

Techniques: Inhibition, Western Blot, Control

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: In some assays recombinant human CK2β protein (GeneTex Inc, Irvine, CA) was added.

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

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: In some assays recombinant human CK2β protein (GeneTex Inc, Irvine, CA) was added.

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

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: In some assays recombinant human CK2β protein (GeneTex Inc, Irvine, CA) was added.

Techniques: Injection, MANN-WHITNEY, Ex Vivo, Control

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: In some assays recombinant human CK2β protein (GeneTex Inc, Irvine, CA) was added.

Techniques: Expressing, Western Blot, Injection