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

Journal: Human Genetics and Genomics Advances

Article Title: De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway

doi: 10.1016/j.xhgg.2022.100111

Figure Lengend Snippet: CSNK2B variants identified in IDCS patients (A) Upper panel: genomic structure of human CSNK2B . The seven exons of CSNK2B are displayed by boxes, which are drawn to scale (1 kb = 1 cm). The filled boxes depict the open reading frame, and the open boxes show the UTRs. The introns are drawn as connecting lines of arbitrary length. Vertical lines on filled boxes indicate the positions of the variants. Lower panel: 215-amino-acid-long CK2β protein composed of five domains along with identified variants are shown. The protein structure is constructed according to the indicated scale bar. Figure is recreated from Bibby and Litchfield (2005). (B) Cross-species alignment showing conservation of Asp32 of CK2β. Note that Asp32 is conserved in all species. Asterisk (∗) is used for the conserved residues, colon (:) for conservative changes, and dot (.) for semiconservative changes. (C) Structural overview of the CK2α2β2 holoenzyme along with a modeled substrate peptide and the ATP analogue AMPPNP. Dotted circle shows the wild-type amino acid Asp32 and substrate peptide in its close proximity. (D) Zoomed picture focuses on the critical neighborhood around Asp32 of CK2β illustrating the IDCS-associated variants Asp32Asn and Asp32His. Two sulfate ions visible in the human CK2α structure PDB: 2PVR were drawn in ball-and-sticks representation; as outlined in the section, these sulfate ions served as an orientation to model the p+1 and p+3 side chains of the substrate peptide DDSDDD (blue carbon atoms) into the active site.

Article Snippet: The following primary antibodies were used for IF and IB: rabbit polyclonal CK2β (Ab76025 for IF; Abcam), mouse monoclonal CK2β (Sc-12739 for IB; Santa Cruz Biotechnologies), mouse monoclonal β-catenin (05-665, active form dephosphorylated on Ser33, Ser37, and T41; Millipore), , rabbit monoclonal β-catenin (ab32572, non-active form phosphorylated on Ser33, Ser37, and T41; Abcam), , rabbit monoclonal DVL3 (ab76081; Abcam), mouse monoclonal α-tubulin (T8328; Sigma-Aldrich), rabbit polyclonal Lamin A/C (H-110, sc-20681; Santa Cruz Biotechnology), mouse monoclonal GAPDH peroxidase-conjugated (G8795 for IB; Sigma), mouse monoclonal anti-GFP (K3-184-2), and in-house manufactured mouse monoclonal glutathione S-transferase (GST).

Techniques: Construct

Journal: Human Genetics and Genomics Advances

Article Title: De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway

doi: 10.1016/j.xhgg.2022.100111

Figure Lengend Snippet: Quantification of CSNK2B transcript and encoded protein product along with cellular localization in LCLs (A) Quantitative real-time PCR data showing 3-fold increased expression of CSNK2B mRNA in patient (GenBank: NM_001320.7:c.94G>C (p.Asp32His)) as compared with wild type. CSNK2B mRNA amounts were quantified relative to control sample. ∗∗∗p = 0.0001 calculated by Student’s t test. n = 3; error bars represent standard deviation (SD). (B) Graph shows 2-fold increased expression of CSNK2B mRNA of subject (GenBank: NM_001320.7 :c.94G>A (p.Asp32Asn)) resulting from amplifying a second set of primers as compared with wild type. ∗∗∗p = 0.0001 calculated by Student’s t test. n = 3; error bars represent SD. (C) Confocal microscopy images showing increased amount of CK2β (green) in nuclei and in cytoplasm of subject-derived (NP_001311.3; p.Asp32His and NP_001311.3; p.Asp32Asn) LCLs as compared with wild type. GM130 (red) serves as marker of Golgi apparatus, and DAPI (blue) indicates staining of nucleus. Scale bar: 10 μm. (D) Immunoblotting shows an increased amount of CK2β in whole-cell lysates obtained from mutant (NP_001311.3; p.Asp32His and NP_001311.3; p.Asp32Asn) LCLs versus wild type. α-Tubulin is used as loading controls. (E) Immunoblots show an increased amount of CK2β in cytosolic and nuclear fraction in NP_001311.3; p.Asp32His mutant versus wild type. Loading controls are α-tubulin and lamin A/C, for cytosol and nucleus, respectively.

Article Snippet: The following primary antibodies were used for IF and IB: rabbit polyclonal CK2β (Ab76025 for IF; Abcam), mouse monoclonal CK2β (Sc-12739 for IB; Santa Cruz Biotechnologies), mouse monoclonal β-catenin (05-665, active form dephosphorylated on Ser33, Ser37, and T41; Millipore), , rabbit monoclonal β-catenin (ab32572, non-active form phosphorylated on Ser33, Ser37, and T41; Abcam), , rabbit monoclonal DVL3 (ab76081; Abcam), mouse monoclonal α-tubulin (T8328; Sigma-Aldrich), rabbit polyclonal Lamin A/C (H-110, sc-20681; Santa Cruz Biotechnology), mouse monoclonal GAPDH peroxidase-conjugated (G8795 for IB; Sigma), mouse monoclonal anti-GFP (K3-184-2), and in-house manufactured mouse monoclonal glutathione S-transferase (GST).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Standard Deviation, Confocal Microscopy, Derivative Assay, Marker, Staining, Western Blot, Mutagenesis

Journal: Human Genetics and Genomics Advances

Article Title: De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway

doi: 10.1016/j.xhgg.2022.100111

Figure Lengend Snippet: Interaction of CK2 subunits and kinase activity of mutated beta subunits (A) Graph shows interaction of CK2 subunits analyzed by microscale thermophoresis (MST). Graph in the left panel shows K D value (12.9 ± 3.9 nM) in wild-type CK2β bound to CK2α239 Cy5 . n = 3. Graph in the right panel shows K D values (15.8 ± 5.3 nM) in mutant CK2β (NP_001311.3; p.Asp32His) bound to CK2α239 Cy5 . n = 5. (B) Graph shows the amount of phosphorylated substrate peptide in the given time from capillary electrophoresis assay. Note that CK2 containing CK2β: NP_001311.3; p.Asp32His shows decreased amount of phosphorylated peptide as compared with wild type, whereas CK2 containing NP_001311.3; p.Asp32Asn shows no significant differences. The color and shape key denoting all the samples given in the figure. n = 3; error bars represent SD. (C) Kinase activity of CK2 wild type and mutants based on capillary electrophoresis assay. Bar graph shows kinase activity of CK2 containing wild-type and mutant beta subunits (NP_001311.3; p.Asp32His and NP_001311.3; p.Asp32Asn). Note that the activity of CK2 containing NP_001311.3; p.Asp32His is reduced as compared with wild type, but CK2 containing NP_001311.3; p.Asp32Asn variant does not show any significant difference. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ns p ˃ 0.05 (Student’s t test). n = 3; error bars represent SD. ns, non-significant.

Article Snippet: The following primary antibodies were used for IF and IB: rabbit polyclonal CK2β (Ab76025 for IF; Abcam), mouse monoclonal CK2β (Sc-12739 for IB; Santa Cruz Biotechnologies), mouse monoclonal β-catenin (05-665, active form dephosphorylated on Ser33, Ser37, and T41; Millipore), , rabbit monoclonal β-catenin (ab32572, non-active form phosphorylated on Ser33, Ser37, and T41; Abcam), , rabbit monoclonal DVL3 (ab76081; Abcam), mouse monoclonal α-tubulin (T8328; Sigma-Aldrich), rabbit polyclonal Lamin A/C (H-110, sc-20681; Santa Cruz Biotechnology), mouse monoclonal GAPDH peroxidase-conjugated (G8795 for IB; Sigma), mouse monoclonal anti-GFP (K3-184-2), and in-house manufactured mouse monoclonal glutathione S-transferase (GST).

Techniques: Activity Assay, Microscale Thermophoresis, Mutagenesis, Electrophoresis, Variant Assay

Journal: Human Genetics and Genomics Advances

Article Title: De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway

doi: 10.1016/j.xhgg.2022.100111

Figure Lengend Snippet: Impacts of variants on the dynamics of β-catenin and DVL3 (A) Pull-down assay from HeLa total protein extracts indicates reduced interaction of endogenous β-catenin with mutant (NP_001311.3; p.Asp32His) GST-tagged CK2β as compared with wild type. GST serves as negative control. Bands of approximately 85 kDa of β-catenin were observed on western blot after probing with rabbit monoclonal β-catenin antibody. GST-fused proteins were visualized by probing the membrane with in-house-generated mouse monoclonal GST antibody. (B) Pull-down assay shows reduced interaction of DVL3 with GST-fused CK2β mutant (NP_001311.3; p.Asp32His) as compared with wild-type. GST was used as negative control. Bands of approximately 78 kDa of DVL3 were observed by rabbit monoclonal DVL3 antibody. (C) Immunofluorescence shows decreased amount of active β-catenin (red) in nuclei of CK2β: NP_001311.3; p.Asp32His (left panel) and CK2β: NP_001311.3; p.Asp32Asn (right panel) LCLs as compared with the wild type. Localization pattern of non-active β-catenin (green) remains the same in wild-type and both mutant LCLs. DAPI (blue) indicates staining of nucleus. Scale bar, 5 μm (left panel); 10 μm (right panel). (D) Graph showing reduced kinase activity of CK2 carrying mutants (NP_001311.3; p.Asp32His and NP_001311.3; p.Asp32Asn) of CK2β as compared with wild type measured by ADP-Glo assay. Note that β-catenin was used as substrate. Error bars represent SD; n = 3. ∗∗p ≤ 0.01 (Student’s t test).

Article Snippet: The following primary antibodies were used for IF and IB: rabbit polyclonal CK2β (Ab76025 for IF; Abcam), mouse monoclonal CK2β (Sc-12739 for IB; Santa Cruz Biotechnologies), mouse monoclonal β-catenin (05-665, active form dephosphorylated on Ser33, Ser37, and T41; Millipore), , rabbit monoclonal β-catenin (ab32572, non-active form phosphorylated on Ser33, Ser37, and T41; Abcam), , rabbit monoclonal DVL3 (ab76081; Abcam), mouse monoclonal α-tubulin (T8328; Sigma-Aldrich), rabbit polyclonal Lamin A/C (H-110, sc-20681; Santa Cruz Biotechnology), mouse monoclonal GAPDH peroxidase-conjugated (G8795 for IB; Sigma), mouse monoclonal anti-GFP (K3-184-2), and in-house manufactured mouse monoclonal glutathione S-transferase (GST).

Techniques: Pull Down Assay, Mutagenesis, Negative Control, Western Blot, Membrane, Generated, Immunofluorescence, Staining, Activity Assay, Glo Assay

Journal: Human Genetics and Genomics Advances

Article Title: De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway

doi: 10.1016/j.xhgg.2022.100111

Figure Lengend Snippet: Whole-phosphoproteome profiling of patient LCLs (NP_001311.3; p.Asp32His) (A) Venn diagram showing whole phosphoproteome of mutant (NP_001311.3; p.Asp32His) and wild-type LCLs. Note that among 1,347 (green) proteins, 425 (pink) are non-phosphorylated. These 425 contain 313 putative CK2 substrates motifs (shown in gray). The FC is >2; q value is 0.05. (B) Venn diagram showing whole phosphoproteome of NP_001311.3; p.Asp32His mutant and wild-type LCLs. Note that among 1,347 proteins shown in gray, 379 are hyper-phosphorylated (shown in green). These 379 contain 231 substrates of CK2 (shown in pink). The FC is >2; q value is 0.05. (C) Donut graph generated by Funrich shows the percent of genes involved in a biological pathway. Note that each pathway is color coded and the key is given. (D) The diagram presents substrate motifs of different kinases that are hyper-phosphorylated (shown in green) and hypo-phosphorylated (shown in pink). The respective p values of enriched motifs are written corresponding to each kinase.

Article Snippet: The following primary antibodies were used for IF and IB: rabbit polyclonal CK2β (Ab76025 for IF; Abcam), mouse monoclonal CK2β (Sc-12739 for IB; Santa Cruz Biotechnologies), mouse monoclonal β-catenin (05-665, active form dephosphorylated on Ser33, Ser37, and T41; Millipore), , rabbit monoclonal β-catenin (ab32572, non-active form phosphorylated on Ser33, Ser37, and T41; Abcam), , rabbit monoclonal DVL3 (ab76081; Abcam), mouse monoclonal α-tubulin (T8328; Sigma-Aldrich), rabbit polyclonal Lamin A/C (H-110, sc-20681; Santa Cruz Biotechnology), mouse monoclonal GAPDH peroxidase-conjugated (G8795 for IB; Sigma), mouse monoclonal anti-GFP (K3-184-2), and in-house manufactured mouse monoclonal glutathione S-transferase (GST).

Techniques: Mutagenesis, Generated

Journal: Human Genetics and Genomics Advances

Article Title: De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway

doi: 10.1016/j.xhgg.2022.100111

Figure Lengend Snippet: CK2 is the crucial member of the canonical Wnt signaling pathway Mutant CK2 has reduced the phosphorylation rate of β-catenin, which thus fails to translocate to the nucleus and consequently affects the regulation of Wnt target genes. This disruption of Wnt signaling leads to the phenotype seen in the studied patient.

Article Snippet: The following primary antibodies were used for IF and IB: rabbit polyclonal CK2β (Ab76025 for IF; Abcam), mouse monoclonal CK2β (Sc-12739 for IB; Santa Cruz Biotechnologies), mouse monoclonal β-catenin (05-665, active form dephosphorylated on Ser33, Ser37, and T41; Millipore), , rabbit monoclonal β-catenin (ab32572, non-active form phosphorylated on Ser33, Ser37, and T41; Abcam), , rabbit monoclonal DVL3 (ab76081; Abcam), mouse monoclonal α-tubulin (T8328; Sigma-Aldrich), rabbit polyclonal Lamin A/C (H-110, sc-20681; Santa Cruz Biotechnology), mouse monoclonal GAPDH peroxidase-conjugated (G8795 for IB; Sigma), mouse monoclonal anti-GFP (K3-184-2), and in-house manufactured mouse monoclonal glutathione S-transferase (GST).

Techniques: Mutagenesis, Phospho-proteomics, Disruption

Journal: Journal of Biological Rhythms

Article Title: Casein Kinase 1 Underlies Temperature Compensation of Circadian Rhythms in Human Red Blood Cells

doi: 10.1177/0748730419836370

Figure Lengend Snippet: Casein kinase inhibition alters the circadian rhythm of red blood cells (RBCs). (A) Immunoblots showing the expression profiles of human CK1 and CK2 paralogs in human RBCs and in human U2OS cells. Data from 2 donors are shown. U2OS cells transfected with V5-CK1α , CK1δ-V5, HA-CK1ε , HA-CK2α’ and Myc-CK2β (both ) served as positive controls. (B-D) Dielectrophoresis (DEP) measurements of membrane conductance ( G eff ) in isolated RBCs incubated at constant 37°C over 2 days in the presence of 100 nM TTP22 (CK2 inhibitor) (B), 1 μM D4476 (CK1 inhibitor) (C), or 3 μM PF-670462 (highly potent CK1 inhibitor) (D) versus vehicle control (mean ± SEM, n = 4. Extra sum-of-squares F test p values reported from non-linear regression comparison of fits, H 1 = damped cosine model, H 0 = straight line). (E) The period of G eff rhythms are significantly affected by inhibition of CK1 and CK2 (period values derived from best fit cosinor ± SEM, n = 4. Extra sum-of-squares F test p values reported following comparison of best fit cosinor . ** p < 0.01, * p < 0.05).

Article Snippet: Primary antibodies were as follows: rabbit anti-CK1α (1:1000, ab108296; Abcam, Cambridge, UK), mouse anti-CK1δ (1:1000, ab85320; Abcam), mouse anti-CK1ε (1:2000, ab82426; Abcam), rabbit anti-CK2α‘ (1:1000, ab10474; Abcam) and rabbit anti-CK2β (1:1000, ab133576; Abcam).

Techniques: Inhibition, Western Blot, Expressing, Transfection, Isolation, Incubation, Derivative Assay

Journal: Oncotarget

Article Title: Under-expression of CK2β subunit in ccRCC represents a complementary biomarker of p-STAT3 Ser727 that correlates with patient survival

doi: 10.18632/oncotarget.23422

Figure Lengend Snippet: ( A ) Western blot analysis of the epithelial (E-cadherin) and mesenchymal markers (Vimentin, Snail1) in HK-2 and 786-O cell lines silenced for either CK2α or CK2β subunits. Values below each band represent the relative protein expression level respect to their corresponding control cell line (HK-2 or 786-O shCV). ( B ) Effect of CK2α or CK2β-downregulation on the relative expression of HIF1α in HK-2 cells and of HIF2α in 786-O cells. Values below each band represent the relative protein expression level respect to their corresponding control cell line (HK-2 or 786-O shCV).

Article Snippet: Primary antibodies used were: anti-CK2α H-286 (sc-9030, Santa Cruz Biotechnology), anti-CK2α 1AD9 (05-1431, Millipore), anti-CK2α’ (A300-199A, Bethyl Laboratories), anti-CK2β 6D5 (sc-12739, Santa Cruz Biotechnology), anti-CK2β(h) (raised in rabbits immunized with whole human recombinant CK2β as described in [ ]), anti-p-Akt1 Ser129 (ab133458, EPR6150, Abcam), anti-Akt1 (2938, C73H10, Cell Signaling), Anti-Akt2 (3063, D6G4, Cell Signaling), anti-E-Cadherin (610181, BD Biosciences), anti-Snail1 (3895, L70G2, Cell Signaling), anti-Vimentin (V6389 Sigma-Aldrich), anti-p-STAT3 Tyr705 (4113, M9C6, Cell Signaling), anti-p-STAT3 Ser727(9134, Cell Signaling), anti-STAT3 (9139, 124H6, Cell Signaling), anti-HIF-1α (ab82832, Abcam), anti-HIF-2α (ab199, Abcam), anti-β-actin (sc-47778, Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: Under-expression of CK2β subunit in ccRCC represents a complementary biomarker of p-STAT3 Ser727 that correlates with patient survival

doi: 10.18632/oncotarget.23422

Figure Lengend Snippet: ( A ) Western blot analysis of CK2α, CK2α’ and CK2β expression levels in HK-2 and 786-O cells lines silenced for either CK2α or CK2β subunits. The type of antibody used to detect each CK2 subunit is indicated in parenthesis. Values below each band correspond to the relative subunit expression level respect to their corresponding control cell line (either HK-2 shCV or 786-O shCV). ( B ) In-gel CK2 activity assay with β-casein as substrate. ( C ) CK2 activity of silenced cell lines using either the CK2-tide (phosphorylated by the holoenzyme and catalytic subunits) or the eIF2β-tide (phosphorylated by CK2 holoenzyme). Data are represented as mean +/- SEM. ( D ) Effects of CK2α or CK2β-downregulation on Akt1 phosphorylation at Ser129. Akt1 phosphorylation at its Ser129 (p-Akt S129) and total Akt1 and Akt2 levels were detected in cell extracts from HK-2 and 786-O cells lines silenced for either CK2α or CK2β subunits by western blot using specific antibodies. Values below each band correspond to the relative protein expression level respect to their corresponding control cell line (HK-2 shCV and 786-O shCV).

Article Snippet: Primary antibodies used were: anti-CK2α H-286 (sc-9030, Santa Cruz Biotechnology), anti-CK2α 1AD9 (05-1431, Millipore), anti-CK2α’ (A300-199A, Bethyl Laboratories), anti-CK2β 6D5 (sc-12739, Santa Cruz Biotechnology), anti-CK2β(h) (raised in rabbits immunized with whole human recombinant CK2β as described in [ ]), anti-p-Akt1 Ser129 (ab133458, EPR6150, Abcam), anti-Akt1 (2938, C73H10, Cell Signaling), Anti-Akt2 (3063, D6G4, Cell Signaling), anti-E-Cadherin (610181, BD Biosciences), anti-Snail1 (3895, L70G2, Cell Signaling), anti-Vimentin (V6389 Sigma-Aldrich), anti-p-STAT3 Tyr705 (4113, M9C6, Cell Signaling), anti-p-STAT3 Ser727(9134, Cell Signaling), anti-STAT3 (9139, 124H6, Cell Signaling), anti-HIF-1α (ab82832, Abcam), anti-HIF-2α (ab199, Abcam), anti-β-actin (sc-47778, Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Activity Assay