Journal: bioRxiv

Article Title: Phosphorylation of the rod-tail hinge region of cingulin regulates its interaction with nonmuscle myosin-2B

doi: 10.64898/2026.04.02.716052

Figure Lengend Snippet: Inhibition of either CK1 or CK2 promotes the rescue of junctional NM2B in cingulin-KO cells expressing the depho-6 mutant of cingulin. (A) Top: scheme of GFP-tagged canine CGN (cCGN-FL), with the GFP tag (green), globular head (gray), coiled-coil rod (white) and globular tail (blue) domains (amino-acid residue boundaries are indicated above the scheme). Bottom: C-terminal sequences of WT canine CGN (cCGN-FL, sequence 1140–1190 with specific residues indicated in the region) and corresponding sequences of the cCGN-dephosphomimetic-6 mutant (depho-6), which does not rescue junctional NM2B (Fig. S1K’). (B-G) IF microscopy analysis and localization (left) and quantification of junctional labeling (relative fluorescence intensity) (right) of NM2B in CGN-KO MDCK cells rescued with GFP-cCGN-FL (B, D and F), or with GFP-cCGN-depho-6 mutant (C, E and G) treated either with DMSO (B and C) or with CK1 inhibitor (D and E: 25 μM, 8h) or with CK2 inhibitor (F and G: 25 μM, 14h). Arrows and arrowheads show increased, normal and decreased/undetected junctional labeling, respectively. Quantifications of relative fluorescent intensity (RFI) shows the ratio between the junctional staining of NM2B versus the junctional marker PLEKHA6 (n=70 junctions) from three independent experiments. Data in quantifications are represented as mean±SD. Statistical significance was determined by unpaired Mann-Whitney’s test. ***p≤0.001. Scale bar (G)= 10 μm.

Article Snippet: Drugs treatments were as follows (final concentration, duration, catalog number and source): CK1 inhibitor (25 μM, 8 h, D4476 MedChemExpress) and CK2 inhibitor (5 μM, 14 h, CX4945 Selleckchem).

Techniques: Inhibition, Expressing, Mutagenesis, Residue, Sequencing, Microscopy, Labeling, Fluorescence, Staining, Marker

Journal: Nutrients

Article Title: Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin

doi: 10.3390/nu10091148

Figure Lengend Snippet: Early phosphorylation of p53 in S20 induced by Resv is necessary for p53-stability in MCF-7 R cells. ( A ) MCF-7 R cells were treated with CDDP (6 μM) with or without Resv (100 μM) and ( B ) MCF-7 cells were treated with Resv (100 μM); both cell cultures were treated for 6 h with specific p53-pS20 site kinase inhibitors: CK1 (60 μM), CHK2 (25 μM) or AMPK (40 μM). Total and phospho-p53 contents are assessed by Western blot using antibodies directed against total p53 (DO-1) or against the specific phosphorylated residue on S20, as indicated. ( C ) MCF-7 and MCF-7 R cells were treated with a DMSO–ethanol vehicle as control or CDDP (6 μM) with resveratrol (100 μM) and cultured in combination with CK1 (60 μM), CHK2 (25 μM) or AMPK (40 μM) inhibitors for 48 h and were double-stained with Annexin V and propidium iodide (PI) followed by flow cytometry analysis to determine apoptotic cells. The viable cells are located in the lower left quadrant (double negative with Annexin V–/PI–). Apoptotic cells (Annexin V+/PI–) appear in the lower right (early apoptosis) and upper right (late apoptosis) quadrant of data plots. Data are presented as a percentage of the cell population. ( D ) The combined results of three independent cytometry analyses depicting the mean levels of total apoptotic cells are shown. Results are presented as the means ± SD. *** p < 0.001 by one-way ANOVA followed by Turkey’s Multiple Comparison test.

Article Snippet: The AMPK inhibitor Compound C (or dorsomorphin), the CK1 inhibitor D4476, the Chk2 inhibitor, anti-rabbit and anti-mouse secondary antibodies, mouse monoclonal anti-phospho-ATM (S1981), rabbit polyclonal anti-ATM, monoclonal anti-p53-HRP (DO-1), and monoclonal anti-BCL-2 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: Phospho-proteomics, Western Blot, Residue, Control, Cell Culture, Staining, Flow Cytometry, Cytometry, Comparison

Journal: Nutrients

Article Title: Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin

doi: 10.3390/nu10091148

Figure Lengend Snippet: In the MCF-7 resistant cell variant (MCF-7 R ), Resv attenuates phosphorylation in S15 and S46 of p53 by dephosphorylation and deactivation of ATM. However, it activates kinases CK1, CHK2, and AMPK to induce phosphorylation of p53 in S20 (which is required to activate p53 in order to upregulate BAX and PUMA genes) and modifies the ratio between BCL-2/BAX expression. The BAX protein was increased while BCL-2 protein was decreased, restoring apoptosis and overcoming chemoresistance. On the other hand, the overexpression of BCL-2 in MCF-7 R cells after CDDP treatment maintains the chemoresistance and blocks apoptosis despite the phosphorylation of p53 in S15 and S46 and the upregulation of NOXA and PUMA .

Article Snippet: The AMPK inhibitor Compound C (or dorsomorphin), the CK1 inhibitor D4476, the Chk2 inhibitor, anti-rabbit and anti-mouse secondary antibodies, mouse monoclonal anti-phospho-ATM (S1981), rabbit polyclonal anti-ATM, monoclonal anti-p53-HRP (DO-1), and monoclonal anti-BCL-2 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: Variant Assay, Phospho-proteomics, De-Phosphorylation Assay, Expressing, Over Expression

Journal: PLoS Pathogens

Article Title: Pathogen Recognition Receptor Signaling Accelerates Phosphorylation-Dependent Degradation of IFNAR1

doi: 10.1371/journal.ppat.1002065

Figure Lengend Snippet: (A) KR-2 cells were infected with HSV (MOI 0.1) and were treated with either DMSO (control) or CK1 inhibitor D4476 (12.5 µM for 1 h) before harvesting at 0, 21, or 23 h post-infection. Cell lysates were immunoprecipitated with the IFNAR1 antibody and analyzed by IB with the indicated antibodies. (B) KR-2 cells were infected with HSV (MOI 0.1) and were harvested at 22 or 24 h post-infection. Cell lysates were analyzed by IFNAR1 IP followed by IB using antibodies against ubiquitin, phospho-S532, and IFNAR1. (C) KR-2 cells expressing Flag-IFNAR1 (wild type or S532A mutant) were infected with HSV (MOI 0.1) and harvested 24 h post-infection. The phosphorylation and ubiquitination of Flag-IFNAR1 were analyzed by IP using anti-Flag antibodies followed by an IB analysis as described in panel D. (D) KR-2 cells expressing the indicated Flag-IFNAR1 (wild type or S532A mutant) were infected with HSV (MOI 0.1) and harvested 30 h post-infection. Levels of exogenous IFNAR1 and β-actin were analyzed by IB using the indicated antibodies.

Article Snippet: CK1 inhibitor D4476 was from Tocris.

Techniques: Infection, Control, Immunoprecipitation, Ubiquitin Proteomics, Expressing, Mutagenesis, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: Pathogen Recognition Receptor Signaling Accelerates Phosphorylation-Dependent Degradation of IFNAR1

doi: 10.1371/journal.ppat.1002065

Figure Lengend Snippet: (A) Mouse bone marrow-derived macrophages (BMM) were pre-treated with LPS (5 µg/mL for 2.5 h) in the presence of either IFNα/β neutralizing antibodies (1000 U/mL) or the p38 kinase inhibitor SB203580 (10 µM) or both, as indicated. Cells were then washed and incubated with murine IFNβ (250 IU/mL for 30 min). The phosphorylation and levels of STAT1 are analyzed in . The asterisk denotes an experiment where neutralizing antibodies were added together with IFNβ. (B) BMM from wild type mice or mice expressing the IFNAR1 S526A mutant were treated and analyzed as indicated according to the description in panel A. (C) Mouse bone marrow-derived dendritic cells (BMDC) from wild type or IFNAR1-null mice were untreated or pre-treated with VX-702 (1 µM for 1 h) and then activated with LPS (2 µg/mL for 48 h). The percent of CD11c-positive and PI-negative viable DCs is depicted as an average of three independent experiments (± S.D.). The asterisk denotes p<0.05 in the t -test. (D) BMDC from wild type mice were untreated or pre-treated with VX-702 (1 µM for 1 h) and then treated with a neutralizing anti-IFNAR1 antibody (MAR, 10 µg/mL) or normal mouse IgG (mIgG) and activated by LPS (2 µg/mL for 48 h). The percent of CD11c-positive and PI-negative viable DCs is depicted as an average of three independent experiments (± S.D.). The asterisk denotes p<0.05 in the t -test. (E) Depicted is a hypothetical model that reflects a dual mode of regulation of the IFNα/β responses by PRR signaling (gray blunt arrows represent the inhibitory effects). The ligand-inducible pathway (that involves activation of TYK2 and PKD2) limits the extent of IFNα/β signaling in a cell that has been already exposed to these cytokines. Conversely, the ligand/JAK-independent pathway may (that involves PERK-dependent or independent activation of p38 kinase, priming phosphorylation and ensuing phosphorylation of IFNAR1 degron by CK1α) render a naïve cell less sensitive to its future encounters with Type I IFNs. The activation of PRR both induces production of endogenous Type I IFN and downregulates IFNAR1 in the very same IFN-producing cells. This mechanism may support the viability of the IFN-producing cells and temper an overall activity of IFNα/β pathways.

Article Snippet: CK1 inhibitor D4476 was from Tocris.

Techniques: Derivative Assay, Incubation, Phospho-proteomics, Expressing, Mutagenesis, Activation Assay, Activity Assay

Journal: Aging

Article Title: The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway

doi:

Figure Lengend Snippet: ( A ) Linear peptide docking sites for enzymes that regulate p53 function. The N-terminus is composed of three transactivation motifs,TAD1, TAD2, and Proline-repeat domain (PRD). A key regulatory domain in the C-terminus (REG) contains the acetylation motifs and phosphorylation site and flanks the Tetramerization domain (TET). The overlapping, but distinct, linear polypeptide docking motifs for p53 regulators include the acetyltransferase p300, the E3 ubiquitin ligase MDM2, iASPP, and the protein kinases including CDK, CK2, CK1, and CHK2 are highlighted. ( B ) Conservation of key phospho-acceptor sites between urochordate and human. The panel highlights the conservation of amino acids and phospho-acceptor sites in the BOX-I transactivation domain of p53 (TAD1 in Figure ) between human and urochordate ( Ciona intestinalis ). The ATM phospho-acceptor site at Ser15 and the Calcium Calmodulin kinase/CK1 phospho-acceptors sites at Thr18 and Ser20 are highlighted as indicated.

Article Snippet: The AMPK inhibitor Compound C (or Dorsomorphin), the CHK1 inhibitor SB218078, the CHK2 inhibitor, and the CK1 inhibitor D4476 were purchased from Merck Chemicals (Nottingham, UK).

Techniques: Phospho-proteomics, Ubiquitin Proteomics

Journal: Aging

Article Title: The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway

doi:

Figure Lengend Snippet: p53 is activated by distinct stresses, some of which include as indicated, ionising radiation, viral infection, metabolic stress induced by an altered AMP/ATP ratio, and oncogene activation. The X-ray-induced Ser20 site kinase is ATM-dependent, but its identity is unknown (highlighted by "?"). CK1 is the DNA virus HHV-6B-induced p53 Ser20 kinase, but the upstream sensor is currently undefined (highlighted by "?"). The Ser20 site kinase induced by an elevated AMP/ATP ratio is AMPK, and LKB is the likely upstream sensor. DAPK-1 is the p53 Ser20 kinase induced by inappropriate oncogene activation, and ERK or ARF are the likely upstream sensors. These data support the formation of a model suggesting that the phosphorylation of p53 at Ser20 is triggered by distinct stress-responsive signaling cascades. Future analysis will be required to determine the identity of all the enzymes that mediate stress-induced phosphorylation at this site and "integrate" the p53 response and developing disease models that deregulate these signaling cascades.

Article Snippet: The AMPK inhibitor Compound C (or Dorsomorphin), the CHK1 inhibitor SB218078, the CHK2 inhibitor, and the CK1 inhibitor D4476 were purchased from Merck Chemicals (Nottingham, UK).

Techniques: Infection, Activation Assay, Virus, Phospho-proteomics

Journal: Aging

Article Title: The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway

doi:

Figure Lengend Snippet: ( A ) A CK1 inhibitor attenuates Ser20 site phosphorylation of p53 and p53 induction mediated by HHV-6B infection. MOLT-3 cells were infected with (even-numbered lanes) or without (odd-numbered lanes) HHV-6B for 48 hours in the presence of increasing concentrations (10-100μM) of the CK1 inhibitor D4476 (lanes 3-14) or a DMSO solvent control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. ( B ) A CK1 inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations (10-60μM) of the CK1 inhibitor D4476 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins.

Article Snippet: The AMPK inhibitor Compound C (or Dorsomorphin), the CHK1 inhibitor SB218078, the CHK2 inhibitor, and the CK1 inhibitor D4476 were purchased from Merck Chemicals (Nottingham, UK).

Techniques: Phospho-proteomics, Infection, Solvent, Control, Western Blot, Cell Culture

Journal: Aging

Article Title: The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway

doi:

Figure Lengend Snippet: ( A ) An AMPK inhibitor attenuates Ser20 site phosphorylation of p53 and p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (2.5-20μM) of the AMPK inhibitor Compound C (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. ( B ) An AMPK inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations (1.25-10μM) of the AMPK inhibitor Compound C (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. ( C ) A CK1 inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (10-60μM) of the CK1 inhibitor D4476 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. ( D ) An ATM inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with AICAR. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 0.5mM AICAR for 24 hours after an initial 24-hour pre-treatment with: increasing concentrations (1-10μM) of the ATM inhibitor KU-55933 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins.

Article Snippet: The AMPK inhibitor Compound C (or Dorsomorphin), the CHK1 inhibitor SB218078, the CHK2 inhibitor, and the CK1 inhibitor D4476 were purchased from Merck Chemicals (Nottingham, UK).

Techniques: Phospho-proteomics, Solvent, Control, Western Blot, Cell Culture

Journal: Neoplasia (New York, N.Y.)

Article Title: SCF β-TrCP targets Ajuba for degradation in a GSK3β-dependent manner in colorectal cancer

doi: 10.1016/j.neo.2025.101175

Figure Lengend Snippet: GSK3β promotes ubiquitination and degradation of Ajuba. A Western blot analysis of HCT116 cells treated with CK1 inhibitor D 4476 (50 μM), GSK3β inhibitor CHIR-99021 (50 μM) and S6K1 inhibitor PF-4708671 (50 μM) for 24 h. B HCT116, HT29 and SW480 cells were incubated with GSK3β inhibitor CHIR-99021 (50 μM) for 24 h and collected for western blot. C Western blot analysis for HCT116 cells treated with 50 μM CHIR-99021 at different time points. D HCT116 cells cultured with GSK3β inhibitor CHIR-99021 (50 μM) for 24 h were pretreated with MG132 (20 μM) for 5 h and harvested for IP. E Western blot analysis of HCT116, SW480 and RKO cells transfected with siRNAs targeting GSK3β for 72 h. F Overexpression of Myc-GSK3β and HA-Ajuba to evaluate the expression level of exogenous Ajuba. G HEK293T cells were transfected with increasing amounts of Myc-GSK3β plasmid, followed by western blot analysis. H, I Cells were transfected with the indicated plasmids for 36 h with or without CHIR-99021 (50 μM) for 24 h and exogenous interaction between GSK3β and Ajuba was analyzed with western blot. J, K Upper panel: RKO cells transfected with two GSK3β siRNAs for 72 h were treated with CHX (50 mg/mL) for the indicated times and then were subjected to western blot. Lower panel: Quantification of Ajuba band intensity. L RKO cells were transfected with GSK3β siRNAs and pretreated with MG132 (20 μM) for 5 h. The cell lysates were immunoprecipitated and then subjected to western blot to analyze the ubiquitination level of Ajuba. M GSK3β and β-TrCP siRNAs were either cotransfected or transfected alone into HCT116 cells and the protein level of Ajuba was detected by western blot.

Article Snippet: CK1 inhibitor D 4476 (S7642), GSK3β inhibitor CHIR-99021 (S1263) and S6K1 inhibitor PF-4708671 (S2163) were purchased from Selleck.

Techniques: Ubiquitin Proteomics, Western Blot, Incubation, Cell Culture, Transfection, Over Expression, Expressing, Plasmid Preparation, Immunoprecipitation

Journal: EBioMedicine

Article Title: SPOP promotes ubiquitination and degradation of LATS1 to enhance kidney cancer progression

doi: 10.1016/j.ebiom.2020.102795

Figure Lengend Snippet: CKΙδ promotes the interaction and degradation of LATS1 by SPOP. (a) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (b) IB analysis of WCLs derived from 293T, A498 and 786-O cells incubated with CKΙ inhibitor IC261 (50μM) or D4476 (20μM) before harvesting. (c) IB analysis of WCLs derived from kidney derived 293T cells transfected with CKΙδ siRNA. (d) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T kidney cells transfected with indicated plasmids and treated with 10μM MG132 for 10 hr before harvesting. (e) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (f) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (g) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T cells transfected with indicated plasmids and treated with 10μM MG132 for 10 h before harvesting.

Article Snippet: CK1 inhibitor IC261 (SC-3561) and D4476 (SC-202522) were purchased from Santa Cruz Biotechnology.

Techniques: Derivative Assay, Transfection, Incubation

Journal: Aging

Article Title: The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway

doi:

Figure Lengend Snippet: ( A ) Linear peptide docking sites for enzymes that regulate p53 function. The N-terminus is composed of three transactivation motifs,TAD1, TAD2, and Proline-repeat domain (PRD). A key regulatory domain in the C-terminus (REG) contains the acetylation motifs and phosphorylation site and flanks the Tetramerization domain (TET). The overlapping, but distinct, linear polypeptide docking motifs for p53 regulators include the acetyltransferase p300, the E3 ubiquitin ligase MDM2, iASPP, and the protein kinases including CDK, CK2, CK1, and CHK2 are highlighted. ( B ) Conservation of key phospho-acceptor sites between urochordate and human. The panel highlights the conservation of amino acids and phospho-acceptor sites in the BOX-I transactivation domain of p53 (TAD1 in Figure ) between human and urochordate ( Ciona intestinalis ). The ATM phospho-acceptor site at Ser15 and the Calcium Calmodulin kinase/CK1 phospho-acceptors sites at Thr18 and Ser20 are highlighted as indicated.

Article Snippet: The AMPK inhibitor Compound C (or Dorsomorphin), the CHK1 inhibitor SB218078, the CHK2 inhibitor, and the CK1 inhibitor D4476 were purchased from Merck Chemicals (Nottingham, UK).

Techniques: Phospho-proteomics, Ubiquitin Proteomics

Journal: Aging

Article Title: The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway

doi:

Figure Lengend Snippet: ( A, B ) A CHK2 inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations [1-10μM ( A ) or 10-100μM ( B )] of the CHK2 inhibitor (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. ( C, D ) A CHK1 inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations [1-10μM ( C ) or 5-50μM ( D )] of the CHK1 inhibitor SB218078 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. ( E ) An ATM inhibitor attenuates Ser20 site phosphorylation of p53, but not p53 induction, mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations (1-10μM) of the ATM inhibitor KU-55933 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins.

Article Snippet: The AMPK inhibitor Compound C (or Dorsomorphin), the CHK1 inhibitor SB218078, the CHK2 inhibitor, and the CK1 inhibitor D4476 were purchased from Merck Chemicals (Nottingham, UK).

Techniques: Phospho-proteomics, Cell Culture, Solvent, Control, Western Blot

Journal: Aging

Article Title: The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway

doi:

Figure Lengend Snippet: ( A, B ) A CHK2 inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations [1-10μM ( A ) or 10-100μM ( B )] of the CHK2 inhibitor (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. ( C, D ) A CHK1 inhibitor does not attenuate Ser20 site phosphorylation of p53 nor p53 induction mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations [1-10μM ( C ) or 5-50μM ( D )] of the CHK1 inhibitor SB218078 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins. ( E ) An ATM inhibitor attenuates Ser20 site phosphorylation of p53, but not p53 induction, mediated by treatment with X-rays. MOLT-3 cells were treated with (even-numbered lanes) or without (odd-numbered lanes) 6Gy X-ray and cultured for 4 hours after an initial 44-hour pre-treatment with: increasing concentrations (1-10μM) of the ATM inhibitor KU-55933 (lanes 5-12), a DMSO solvent control (lanes 3-4), or a culture medium control (lanes 1-2). Cell lysates were examined by Western blotting with antibodies against the indicated proteins.

Article Snippet: The AMPK inhibitor Compound C (or Dorsomorphin), the CHK1 inhibitor SB218078, the CHK2 inhibitor, and the CK1 inhibitor D4476 were purchased from Merck Chemicals (Nottingham, UK).

Techniques: Phospho-proteomics, Cell Culture, Solvent, Control, Western Blot

Journal: PLoS ONE

Article Title: Neurite Outgrowth of Mature Retinal Ganglion Cells and PC12 Cells Requires Activity of CK1δ and CK1ε

doi: 10.1371/journal.pone.0020857

Figure Lengend Snippet: ( A ) Kinase activity in PC12 cells. Equal amounts of protein lysates from PC12 cells either untreated (control) or exposed to NGF for 24 h were loaded onto a 1 ml Resource-Q column for ion exchange chromatography and eluted with a linear gradient of increasing NaCl concentration (represented as a solid diagonal line). Fractions were collected and the CK1δ/ε specific kinase activity was determined in each single fraction as described in detail in Material and Methods. Kinase activities (CPM: counts per minute) are represented by open squares (control) or closed triangles (24 h after NGF stimulation). ( B ) The kinase activity in peak fraction 21 of untreated (control) and NGF stimulated PC12 cells (24 h) was significantly reduced by IC261 specifically inhibiting CK1δ and ε in the micromolar range. Phosphate incorporation into GST-p53 1–64 (FP267) was quantified by Cherenkov counting. ( C ) Kinase activity in rat retina. Equal amounts of protein lysates from rat retinal tissue either untreated or treated with ONC+LI were loaded onto a 1 ml Resource-Q column for ion exchange chromatography and eluted with a linear gradient of increasing NaCl concentration (represented as a solid diagonal line). Fractions were collected and the CK1δ/ε specific kinase activity was determined in each single fraction as described in detail in Material and Methods. Kinase activities (CPM: counts per minute) are represented by open squares (control) or closed triangles (ONC+LI). ( D ) The kinase activity in peak fraction 38 of extracts prepared from ONC+LI treated animals was significantly reduced by the CK1δ/ε specific inhibitor IC261. Phosphate incorporation into GST-p53 1–64 (FP267) was quantified by Cherenkov counting.

Article Snippet: After 30 min, medium was exchanged to growth medium containing the CK1-specific inhibitors IC261 (ICOS Corporation, Bothell, USA) or D4476 (Calbiochem, Darmstadt, Germany) or DMSO/transfection reagent (as a negative control).

Techniques: Activity Assay, Control, Ion Exchange Chromatography, Concentration Assay

Journal: PLoS ONE

Article Title: Neurite Outgrowth of Mature Retinal Ganglion Cells and PC12 Cells Requires Activity of CK1δ and CK1ε

doi: 10.1371/journal.pone.0020857

Figure Lengend Snippet: ( A ) PC12 cells were cultured for 3 days without NGF (control) and in the presence of NGF+vehicle or NGF+CKI-7 (50 and 200 µM) or IC261 (0.5, 1.5 and 50 µM). In a concentration dependent manner the average neurite length of NGF stimulated PC12 cells was markedly reduced in the presence of the CK1-specific inhibitors CKI-7 or IC261. Treatment effects: **p<0.01 and ***p<0.001 compared to cells exposed to NGF+vehicle. ( B, C ) LI stimulated RGCs were cultured in the absence (control) or presence of CKI-7 (B; 50, 200 and 800 µM) or IC261 (C; 0.5, 1.5 and 50 µM). Neurite outgrowth was compromised in a concentration dependent manner. The survival of RGCs was only affected in the presence of CKI-7 at 800 µM. Treatment effects compared to control groups: *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: After 30 min, medium was exchanged to growth medium containing the CK1-specific inhibitors IC261 (ICOS Corporation, Bothell, USA) or D4476 (Calbiochem, Darmstadt, Germany) or DMSO/transfection reagent (as a negative control).

Techniques: Cell Culture, Control, Concentration Assay

Journal: PLoS ONE

Article Title: Neurite Outgrowth of Mature Retinal Ganglion Cells and PC12 Cells Requires Activity of CK1δ and CK1ε

doi: 10.1371/journal.pone.0020857

Figure Lengend Snippet: ( A ) LI stimulated RGCs were cultured for 24 h and then exposed to vehicle or different concentrations of the CK1-specific inhibitors IC261 (0.5, 1.5 and 50 µM) or CKI-7 (50 and 200 µM). The average neurite length of RGCs was determined after 24 h (first control) and 48 h, respectively. Treatment effects: ***p<0.001. ( B ) The number of RGCs per well in all groups was not affected by any treatment described in (A). ( C ) LI stimulated RGCs were cultured for 48 h and subsequently monitored in a flow-through chamber for live cell imaging. In the presence of vehicle or inhibitors IC261 (50 µM) or D4476 (5 µM) cells were recorded for 4 h. Retraction of the outgrown neurite after exposure to IC261 and D4476 is shown at 0, 120 and 240 min. Arrowheads indicate the position of the cell body, arrows the position of the growth cone. Scale bar: 50 µm. ( D ) Quantification of neurite retraction of RGCs over time exposed to vehicle (control, closed squares) or inhibitors IC261 (50 µM, open triangles) or D4476 (5 µM, open squares) specifically inhibiting CK1δ and ε.

Article Snippet: After 30 min, medium was exchanged to growth medium containing the CK1-specific inhibitors IC261 (ICOS Corporation, Bothell, USA) or D4476 (Calbiochem, Darmstadt, Germany) or DMSO/transfection reagent (as a negative control).

Techniques: Cell Culture, Control, Live Cell Imaging