ck2 α and β subunits (177 nm ck2α, 4.4 nm ck2 α 2 β 2 )  (Biaffin Inc)

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: Antibodies directed against CK2 α/α′ and β were from Santa Cruz Biotechnology.

Techniques: Binding Assay, Protein Binding

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: Antibodies directed against CK2 α/α′ and β were from Santa Cruz Biotechnology.

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: Antibodies directed against CK2 α/α′ and β were from Santa Cruz Biotechnology.

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: Antibodies directed against CK2 α/α′ and β were from Santa Cruz Biotechnology.

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: Antibodies directed against CK2 α/α′ and β were from Santa Cruz Biotechnology.

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: Antibodies directed against CK2 α/α′ and β were from Santa Cruz Biotechnology.

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: Antibodies directed against CK2 α/α′ and β were from Santa Cruz Biotechnology.

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: Antibodies directed against CK2 α/α′ and β were from Santa Cruz Biotechnology.

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: Antibodies directed against CK2 α/α′ and β were from Santa Cruz Biotechnology.

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: Antibodies directed against CK2 α/α′ and β were from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Injection

Journal: Nature chemical biology

Article Title: Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling

doi: 10.1038/nchembio.2165

Figure Lengend Snippet: ( a ) S. cerevisiae ABC16 strain lacking 16 ABC transporters is susceptible to beauvericin, and was used to select for beauvericin-resistant mutants. Selection experiment was performed as in . Mutations identified by whole genome sequencing of ten resistant mutants are indicated as amino acid changes. All resistant mutants had nonsynonymous mutations in β regulatory subunits of protein kinase CK2 (shown in red). Resistance phenotypes were assessed as in . ( b ) Loss of function of CKB1 or CKB2 confers beauvericin resistance in the ABC16 strain. Deletion of CKB1 or CKB2 in the ABC16 strain confers resistance to beauvericin. Expression of a wild-type allele of CKB1 or CKB2 in the ABC16 strain lacking the corresponding regulatory subunit gene restores beauvericin susceptibility to the level of the parental ABC16 strain; in contrast, resistance is maintained upon expression of the CKB1 E220 * or CKB2 Q225 * alleles. Resistance phenotypes were assessed as in . (a–b) Experiments performed in biological triplicates with technical duplicates.

Article Snippet: The CK2 α and β subunits (177 nM CK2α, 4.4 nM CK2 α 2 β 2 ) used were recombinant human subunits purchased from Biaffin.

Techniques: Selection, Sequencing, Expressing

Journal: Nature chemical biology

Article Title: Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling

doi: 10.1038/nchembio.2165

Figure Lengend Snippet: ( a ) Left, structure of the beauvericin (BEA) biotinylated analog. Right, AP-MS with three buffer conditions in biological duplicates. Circle color indicates absolute spectral count, size indicates relative protein abundance, and edge indicates SAINT false discovery rate (FDR). Excess unmodified beauvericin added as competitor (E1-2x excess, E2-14x excess, and E3-25x excess). ( b ) Beauvericin enhances CK2 activity. Phosphorylation of CK2 target Sic1 monitored in an in vitro assay using protein lysates from the ABC16 strain, adding beauvericin or vehicle to the reaction. Assay in biological duplicates with technical replicates. Error bars represent s.d. * P <0.05 (t-test). ( c ) Beauvericin and rapamycin are antagonistic. Beauvericin suppresses rapamycin antifungal activity against S. cerevisiae (BY4741) and C. albicans (SN95). Checkerboards performed as in , but for 24 hours. Checkerboards performed in biological triplicates with technical duplicates. ( d ) Beauvericin reduces gene induction and repression in response to rapamycin in S. cerevisiae . Transcript levels of treated (0.2 μg/ml rapamycin; 40 μg/ml beauvericin) and untreated cells measured by qRT-PCR and normalized to ACT1 and untreated sample. Error bars represent s.d. among technical triplicates. Assay is representative of biological triplicates. * P <0.05 compared to untreated, ◆ P <0.05 compared to rapamycin treatment (ANOVA). ( e ) Beauvericin inhibits TORC1 activity. Phosphorylation of TORC1 target (Sch9) and TORC2 target (Ypk1) monitored in yor1 Δ grown ± 5 μg/ml beauvericin for four hours before protein extraction and western analysis. Error bars represent s.d. among biological triplicate experiments. * P <0.05 compared to vehicle (unpaired t-test).

Article Snippet: The CK2 α and β subunits (177 nM CK2α, 4.4 nM CK2 α 2 β 2 ) used were recombinant human subunits purchased from Biaffin.

Techniques: Activity Assay, In Vitro, Quantitative RT-PCR, Protein Extraction, Western Blot

Journal: Nature chemical biology

Article Title: Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling

doi: 10.1038/nchembio.2165

Figure Lengend Snippet: ( a ) Beauvericin (BEA) reduces fluconazole-induced calcineurin activation. S. cerevisiae harboring 4xCDRE- lacZ reporter construct ± fluconazole (FLC, 64 μg/ml), ± geldanamycin (GdA, 5.6 μg/ml), FK506 (1.0 μg/ml), or beauvericin (20 μg/ml). Data are mean ± s.d. from technical triplicates and representative of biological replicates. * P <0.05 relative to untreated, ◆ P <0.05 relative to fluconazole treatment (ANOVA, Tukey post-hoc test). ( b ) Beauvericin induces the heat shock response. C. albicans containing HSP70p-lacZ reporter construct were untreated (−), or treated with geldanamycin (10 μM) or beauvericin (13 μM); both induced expression from the HSP70 promoter. Data representation as described in (a). * P <0.05. ( c ) Beauvericin blocks Hog1 activation in a manner that depends on CK2 subunits. Sodium chloride (NaCl) was used to activate Hog1. Beauvericin (2.5 μg/ml) reduced levels of phosphorylated Hog1 in the ABC16 strain; deletion of CKB1 or CKA2 reduced the effect of beauvericin. Bands were quantified using ImageJ, and the ratio of phosphorylated Hog1 to total Hog1 plotted for one biological replicate. Tubulin was used as loading control. A similar trend was observed in biological triplicate experiments . ( d ) Model for beauvericin mechanism of action. Beauvericin inhibits Pdr5 thereby enabling increased azole intracellular accumulation. Beauvericin also inhibits TORC1, thereby activating CK2. Increased CK2 activity has pleiotropic effects including inhibiting the function of Hsp90 and its client protein calcineurin, which regulate azole resistance.

Article Snippet: The CK2 α and β subunits (177 nM CK2α, 4.4 nM CK2 α 2 β 2 ) used were recombinant human subunits purchased from Biaffin.

Techniques: Activation Assay, Construct, Expressing, Activity Assay

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Casein Kinase II Regulates N -Methyl- d -Aspartate Receptor Activity in Spinal Cords and Pain Hypersensitivity Induced by Nerve Injury

doi: 10.1124/jpet.114.215855

Figure Lengend Snippet: Sequences of two CK2 β -specific siRNAs and one control siRNA These siRNAs were conjugated to chitosan and then administered through intrathecal catheters in rats.

Article Snippet: The membrane was blocked for 30 minutes in 5% skim milk in phosphate-buffered saline containing 0.05% Tween-20 and then incubated with goat anti-CK2 α (sc-6476; Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti-CK2 β primary antibody (sc-46666; Santa Cruz Biotechnology) ( Ye et al., 2012 ) overnight at 4°C.

Techniques: Control, Sequencing

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Casein Kinase II Regulates N -Methyl- d -Aspartate Receptor Activity in Spinal Cords and Pain Hypersensitivity Induced by Nerve Injury

doi: 10.1124/jpet.114.215855

Figure Lengend Snippet: CK2 contributes to the increased NMDAR activity of spinal dorsal horn neurons caused by nerve injury. (A and B) Representative recordings (A) and mean changes (B) in the amplitude of evoked NMDAR-EPSCs and AMPAR-EPSCs of lamina II neurons in SNL rat spinal cord slices treated with vehicle (dimethylsulfoxide, n = 16 neurons), 100 μM DRB (n = 10 neurons), or 2 μM TBB (n = 10 neurons). (C and D) Representative recordings (C) and mean changes (D) in the amplitude of evoked NMDAR-EPSCs and AMPAR-EPSCs of lamina II neurons in control rat spinal slices treated with vehicle (n = 13 neurons) or 100 μM DRB (n = 11 neurons). *P < 0.05 compared with the vehicle control group. Error bars represent the S.E.M.

Article Snippet: The membrane was blocked for 30 minutes in 5% skim milk in phosphate-buffered saline containing 0.05% Tween-20 and then incubated with goat anti-CK2 α (sc-6476; Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti-CK2 β primary antibody (sc-46666; Santa Cruz Biotechnology) ( Ye et al., 2012 ) overnight at 4°C.

Techniques: Activity Assay, Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Casein Kinase II Regulates N -Methyl- d -Aspartate Receptor Activity in Spinal Cords and Pain Hypersensitivity Induced by Nerve Injury

doi: 10.1124/jpet.114.215855

Figure Lengend Snippet: CK2 plays a critical role in nerve injury–induced increase in the postsynaptic NMDAR activity of spinal dorsal horn neurons. (A and B) Original traces (A) and mean changes (B) in the NMDAR currents elicited by puff NMDA to lamina II neurons in SNL rat spinal slices treated with vehicle (dimethylsulfoxide, n = 19 neurons) or DRB (n = 23 neurons). (C) Summary data show AMPAR- and NMDAR-mediated mEPSCs of lamina II neurons in spinal cord slices treated with vehicle or DRB in sham control and SNL rats. *P < 0.05 compared with the vehicle control group. Error bars represent the S.E.M.

Article Snippet: The membrane was blocked for 30 minutes in 5% skim milk in phosphate-buffered saline containing 0.05% Tween-20 and then incubated with goat anti-CK2 α (sc-6476; Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti-CK2 β primary antibody (sc-46666; Santa Cruz Biotechnology) ( Ye et al., 2012 ) overnight at 4°C.

Techniques: Activity Assay, Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Casein Kinase II Regulates N -Methyl- d -Aspartate Receptor Activity in Spinal Cords and Pain Hypersensitivity Induced by Nerve Injury

doi: 10.1124/jpet.114.215855

Figure Lengend Snippet: NMDAR activity of spinal dorsal horn neurons is dynamically controlled by CK2 and calcineurin. (A and B) Representative recordings (A) and mean changes (B) in the amplitude of evoked NMDAR-EPSCs and AMPAR-EPSCs of lamina II neurons in control rat spinal slices treated with vehicle (dimethylsulfoxide, n = 13 neurons), 1 μM FK-506 (n = 10 neurons), 0.1 μM okadaic acid (n = 12 neurons), FK-506 plus DRB (n = 10 neurons), or FK-506 plus TBB (n = 10 neurons). (C) Mean changes in the amplitude of evoked NMDAR-EPSCs and AMPAR-EPSCs of lamina II neurons in SNL rat spinal cord slices treated with vehicle (n = 16 neurons) or 1 μM FK-506 (n = 14 neurons). *P < 0.05 compared with the vehicle control group. Error bars represent the S.E.M.

Article Snippet: The membrane was blocked for 30 minutes in 5% skim milk in phosphate-buffered saline containing 0.05% Tween-20 and then incubated with goat anti-CK2 α (sc-6476; Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti-CK2 β primary antibody (sc-46666; Santa Cruz Biotechnology) ( Ye et al., 2012 ) overnight at 4°C.

Techniques: Activity Assay, Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Casein Kinase II Regulates N -Methyl- d -Aspartate Receptor Activity in Spinal Cords and Pain Hypersensitivity Induced by Nerve Injury

doi: 10.1124/jpet.114.215855

Figure Lengend Snippet: Nerve injury increases CK2α and CK2β protein levels in the dorsal spinal cord. (A–C) Immunoblotting gel images (A) and quantification of CK2α (approximately 45 kDa) (B), and CK2β (approximately 25 kDa) (C) protein levels in the dorsal spinal cord ipsilateral to SNL and contralateral (sham) controls (n = 6 rats in each group). The dorsal spinal cord tissues were obtained 3, 7, 14, and 28 days after surgery. The CK2α and CK2β protein amounts were normalized to β-actin (loading controls) in each sample. *P < 0.05 compared with the contralateral control. Error bars represent the S.E.M.

Article Snippet: The membrane was blocked for 30 minutes in 5% skim milk in phosphate-buffered saline containing 0.05% Tween-20 and then incubated with goat anti-CK2 α (sc-6476; Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti-CK2 β primary antibody (sc-46666; Santa Cruz Biotechnology) ( Ye et al., 2012 ) overnight at 4°C.

Techniques: Western Blot, Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Casein Kinase II Regulates N -Methyl- d -Aspartate Receptor Activity in Spinal Cords and Pain Hypersensitivity Induced by Nerve Injury

doi: 10.1124/jpet.114.215855

Figure Lengend Snippet: Inhibition of CK2 and siRNA knockdown of CK2β expression at the spinal level reduces pain hypersensitivity induced by nerve injury. (A and B) Time course of the effects of intrathecal injection of 100, 200, and 500 ng TBB and vehicle (10% dimethylsulfoxide) on the withdrawal threshold measured with von Frey filaments (A) and a pressure stimulus (B) in rats 3 weeks after SNL or sham surgery (n = 7 to 8 rats per group). (C) Quantitative PCR analysis of CK2α and CK2β mRNA levels in the dorsal spinal cord ipsilateral (Ipsi) and contralateral (Cont) to nerve ligation of SNL rats treated with the two CK2β-specific siRNAs and a scramble control siRNA (n = 4 samples in each group). (D) Immunoblotting gel images (top) and mean changes (bottom) show the CK2β protein levels in the dorsal spinal cord of SNL rats treated with the two CK2β-specific siRNAs and a scramble control siRNA (n = 6 samples in each group). (E) Mean changes in the amplitude and ratio of NMDAR-EPSCs to AMPAR-EPSCs of lamina II neurons recorded from SNL rats treated with the scramble control siRNA (n = 11 neurons) or CK2β-specific siRNA (n = 12 neurons). (F and G) Changes in the paw withdrawal threshold measured with von Frey filaments (F) and a pressure stimulus (G) (n = 7 to 8 rats in each group) of SNL rats treated intrathecally with the two CK2β-specific siRNAs and a scramble control siRNA (n = 7 rats in each group). Threshold tests were performed before siRNA injection each day (indicated by arrows). *P < 0.05 compared with the respective predrug control or the control siRNA group. Error bars represent the S.E.M.

Article Snippet: The membrane was blocked for 30 minutes in 5% skim milk in phosphate-buffered saline containing 0.05% Tween-20 and then incubated with goat anti-CK2 α (sc-6476; Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti-CK2 β primary antibody (sc-46666; Santa Cruz Biotechnology) ( Ye et al., 2012 ) overnight at 4°C.

Techniques: Inhibition, Knockdown, Expressing, Injection, Real-time Polymerase Chain Reaction, Ligation, Control, Western Blot