rabbit monoclonal anti phospho chk1 ser 345  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal anti phospho chk1 ser 345
    HCT116, HT-29 and SiHa cancer cells were chronically adapted at pH 6.5 or maintained at pH 7.4 ( A , C , D ) or native HCT116 cells were acutely exposed to acidic pH e ( B ) or treated with 5-FU at the indicated doses to be compared with cancer cells adapted at pH 6.5 ( E , F ). ( A – C ) Representative immunoblots of total and phosphorylated ATM, ATR, <t>CHK1,</t> CHK2. Actin or tubulin was used as loading control, as indicated. ( D ) Bar graph showing the proportion of tetraploid cells. ( E , F ) Representative immunoblots of total and phosphorylated ATM, ATR, CHK1, CHK2. Actin was used as loading control, as indicated. ( G – J ) Cell viability assays in HCT116 ( G , I ) and HT-29 cancer cells ( H , J ) cultured at pH 7.4 or 6.5, and treated with the indicated doses of ATMi AZD0156 ( G , H ) or ATRi elimusertib ( I , J ) for 72 h. Data information: ( A – J ) data represent n = 3 independent biological replicates. ( D , G – J ) Bar graphs represent means ± SD with three biological replicates ( D ) or six technical replicates ( G – J ), and significance was determined using two-way ANOVA with Tukey’s multiple-comparison analysis (ns non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). .
    Rabbit Monoclonal Anti Phospho Chk1 Ser 345, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Tumor acidosis-induced DNA damage response and tetraploidy enhance sensitivity to ATM and ATR inhibitors"

    Article Title: Tumor acidosis-induced DNA damage response and tetraploidy enhance sensitivity to ATM and ATR inhibitors

    Journal: EMBO Reports

    doi: 10.1038/s44319-024-00089-7

    HCT116, HT-29 and SiHa cancer cells were chronically adapted at pH 6.5 or maintained at pH 7.4 ( A , C , D ) or native HCT116 cells were acutely exposed to acidic pH e ( B ) or treated with 5-FU at the indicated doses to be compared with cancer cells adapted at pH 6.5 ( E , F ). ( A – C ) Representative immunoblots of total and phosphorylated ATM, ATR, CHK1, CHK2. Actin or tubulin was used as loading control, as indicated. ( D ) Bar graph showing the proportion of tetraploid cells. ( E , F ) Representative immunoblots of total and phosphorylated ATM, ATR, CHK1, CHK2. Actin was used as loading control, as indicated. ( G – J ) Cell viability assays in HCT116 ( G , I ) and HT-29 cancer cells ( H , J ) cultured at pH 7.4 or 6.5, and treated with the indicated doses of ATMi AZD0156 ( G , H ) or ATRi elimusertib ( I , J ) for 72 h. Data information: ( A – J ) data represent n = 3 independent biological replicates. ( D , G – J ) Bar graphs represent means ± SD with three biological replicates ( D ) or six technical replicates ( G – J ), and significance was determined using two-way ANOVA with Tukey’s multiple-comparison analysis (ns non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). .
    Figure Legend Snippet: HCT116, HT-29 and SiHa cancer cells were chronically adapted at pH 6.5 or maintained at pH 7.4 ( A , C , D ) or native HCT116 cells were acutely exposed to acidic pH e ( B ) or treated with 5-FU at the indicated doses to be compared with cancer cells adapted at pH 6.5 ( E , F ). ( A – C ) Representative immunoblots of total and phosphorylated ATM, ATR, CHK1, CHK2. Actin or tubulin was used as loading control, as indicated. ( D ) Bar graph showing the proportion of tetraploid cells. ( E , F ) Representative immunoblots of total and phosphorylated ATM, ATR, CHK1, CHK2. Actin was used as loading control, as indicated. ( G – J ) Cell viability assays in HCT116 ( G , I ) and HT-29 cancer cells ( H , J ) cultured at pH 7.4 or 6.5, and treated with the indicated doses of ATMi AZD0156 ( G , H ) or ATRi elimusertib ( I , J ) for 72 h. Data information: ( A – J ) data represent n = 3 independent biological replicates. ( D , G – J ) Bar graphs represent means ± SD with three biological replicates ( D ) or six technical replicates ( G – J ), and significance was determined using two-way ANOVA with Tukey’s multiple-comparison analysis (ns non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). .

    Techniques Used: Western Blot, Cell Culture, Comparison

    Reagents and tools.
    Figure Legend Snippet: Reagents and tools.

    Techniques Used: Membrane, Modification, Protease Inhibitor, Software, Bicinchoninic Acid Protein Assay, Viability Assay

    rabbit anti phospho serine 345 chk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti phospho serine 345 chk1
    Myt1 overexpression inhibits mitotic entry by reducing Cdk1 activity in cells treated with checkpoint kinase inhibitors (A) HeLa cells were seeded onto coverslips in the presence or absence of tetracycline (2 µM) for 24 h. Cells were synchronized in G1-S phase by single thymidine block. Following synchronization cells were released into media containing kinase inhibitors or DMSO for 4 h. Cells were stained for DNA and PH3 and analyzed by immunofluorescence microscopy. The percentage of cells positive for PH3 is shown normalized to DMSO/Tet-. Error bars represent SEM. Experiments were repeated three times. Statistical significance was determined by 2-way ANOVA. ***, p < 0.001; **, p = 0.001. (B) Cell lysates were prepared from tetracycline inducible Myt1 expressing HeLa cells. Cells were treated or not with 2 µM of tetracycline 48 h prior to any drug treatment/control: DMSO, Adavosertib (500 nM), PD166285 (500 nM), UCN-01 (1000 nM), and AZD6738 (1000 nM) for 4 h. Cdk1 activity in lysates from cells was assessed in vitro by incubation with GST-PP1Cα (a Cdk1 substrate). Total pT320-PP1Cα peptide and GST levels were determined by immunoblot. (C) Bar graphs show the quantitation of the absolute Cdk1 activity in cells treated with various checkpoint kinase inhibitors. Error bars represent SD. Statistical analysis was done using a 2-way ANOVA. ****, p < 0.0001**, p = 0.0029. (D) Lysates were also evaluated for the levels of pY15-Cdk1, Cdk1, <t>pS345-Chk1,</t> Chk1, overexpressed Myt1 and Tubulin. Quantitation shows the average pS345-Chk1 and pY15-Cdk1 levels relative to their total proteins and normalized to DMSO with no tetracycline addition. Kinase assay was repeated 5 times or more. Western blotting and the immunofluorescence experiments were repeated three times.
    Rabbit Anti Phospho Serine 345 Chk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors"

    Article Title: Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2023.1270542

    Myt1 overexpression inhibits mitotic entry by reducing Cdk1 activity in cells treated with checkpoint kinase inhibitors (A) HeLa cells were seeded onto coverslips in the presence or absence of tetracycline (2 µM) for 24 h. Cells were synchronized in G1-S phase by single thymidine block. Following synchronization cells were released into media containing kinase inhibitors or DMSO for 4 h. Cells were stained for DNA and PH3 and analyzed by immunofluorescence microscopy. The percentage of cells positive for PH3 is shown normalized to DMSO/Tet-. Error bars represent SEM. Experiments were repeated three times. Statistical significance was determined by 2-way ANOVA. ***, p < 0.001; **, p = 0.001. (B) Cell lysates were prepared from tetracycline inducible Myt1 expressing HeLa cells. Cells were treated or not with 2 µM of tetracycline 48 h prior to any drug treatment/control: DMSO, Adavosertib (500 nM), PD166285 (500 nM), UCN-01 (1000 nM), and AZD6738 (1000 nM) for 4 h. Cdk1 activity in lysates from cells was assessed in vitro by incubation with GST-PP1Cα (a Cdk1 substrate). Total pT320-PP1Cα peptide and GST levels were determined by immunoblot. (C) Bar graphs show the quantitation of the absolute Cdk1 activity in cells treated with various checkpoint kinase inhibitors. Error bars represent SD. Statistical analysis was done using a 2-way ANOVA. ****, p < 0.0001**, p = 0.0029. (D) Lysates were also evaluated for the levels of pY15-Cdk1, Cdk1, pS345-Chk1, Chk1, overexpressed Myt1 and Tubulin. Quantitation shows the average pS345-Chk1 and pY15-Cdk1 levels relative to their total proteins and normalized to DMSO with no tetracycline addition. Kinase assay was repeated 5 times or more. Western blotting and the immunofluorescence experiments were repeated three times.
    Figure Legend Snippet: Myt1 overexpression inhibits mitotic entry by reducing Cdk1 activity in cells treated with checkpoint kinase inhibitors (A) HeLa cells were seeded onto coverslips in the presence or absence of tetracycline (2 µM) for 24 h. Cells were synchronized in G1-S phase by single thymidine block. Following synchronization cells were released into media containing kinase inhibitors or DMSO for 4 h. Cells were stained for DNA and PH3 and analyzed by immunofluorescence microscopy. The percentage of cells positive for PH3 is shown normalized to DMSO/Tet-. Error bars represent SEM. Experiments were repeated three times. Statistical significance was determined by 2-way ANOVA. ***, p < 0.001; **, p = 0.001. (B) Cell lysates were prepared from tetracycline inducible Myt1 expressing HeLa cells. Cells were treated or not with 2 µM of tetracycline 48 h prior to any drug treatment/control: DMSO, Adavosertib (500 nM), PD166285 (500 nM), UCN-01 (1000 nM), and AZD6738 (1000 nM) for 4 h. Cdk1 activity in lysates from cells was assessed in vitro by incubation with GST-PP1Cα (a Cdk1 substrate). Total pT320-PP1Cα peptide and GST levels were determined by immunoblot. (C) Bar graphs show the quantitation of the absolute Cdk1 activity in cells treated with various checkpoint kinase inhibitors. Error bars represent SD. Statistical analysis was done using a 2-way ANOVA. ****, p < 0.0001**, p = 0.0029. (D) Lysates were also evaluated for the levels of pY15-Cdk1, Cdk1, pS345-Chk1, Chk1, overexpressed Myt1 and Tubulin. Quantitation shows the average pS345-Chk1 and pY15-Cdk1 levels relative to their total proteins and normalized to DMSO with no tetracycline addition. Kinase assay was repeated 5 times or more. Western blotting and the immunofluorescence experiments were repeated three times.

    Techniques Used: Over Expression, Activity Assay, Blocking Assay, Staining, Immunofluorescence, Microscopy, Expressing, In Vitro, Incubation, Western Blot, Quantitation Assay, Kinase Assay

    DNA damage and cell cycle checkpoint inhibitors and their effect on ectopic Cdk1 activation. (A) In an unperturbed cell, Cdk1 and cyclin B bind in late S-phase. Phosphorylation of Cdk1 on T14 and Y15 (depicted with red circles) by Myt1 and Wee1 during interphase inhibits Cdk1/cyclin B. Cdc25 removes these inhibitory phosphorylations and activates the Cdk1/cyclin B complexes allowing cells to enter mitosis. The high level of Wee1 activity in many cancer cells prevents their premature entry into mitosis and facilitates their survival in the presence of elevated genotoxic stress compared to normal cells. (B) Upon addition of Adavosertib (inhibition of Wee1) or PD166285 (inhibition of Wee1 and Myt1), the balance between inactive and active Cdk1/cyclin B1 complexes is shifted leading to more active Cdk1/cyclin B1 complexes. This increases the chance of premature entry into mitosis and cell death. (C) Overexpression of Myt1 in the presence of Adavosertib or PD166285 counters the drugs’ effect because phosphorylation of Cdk1 on T14 and Y15 by Myt1 increases inactive Cdk1/cyclin B complexes. This results in checkpoint arrest and rescue from cell death even in the presence of these cell cycle checkpoint kinase inhibitors. (D) Damaged DNA or replication stress can induce the activation of the DNA damage checkpoint, which downregulates Cdk1/cyclin B activity via Cdc25. ATR activates the effector kinase Chk1, which phosphorylates and inhibits the Cdc25 phosphatase family. In parallel, ATR can induce the stabilization and transcriptional activity of p53 leading to increased expression of the CDK inhibitor p21; however, this pathway is downregulated in many cancers (due to non-functional p53 status of many cancers) . (E) Upon addition of UCN-01 or AZD6738 (inhibition of Chk1 and ATR), Cdc25 inactivation in the presence of DNA damage is repressed and the balance between inactive and active Cdk1/cyclin B1 complexes is shifted towards more active Cdk1/cyclin B1 complexes. The cells are at elevated risk of premature mitotic entry. Restoration of Cdc25 activity in the presence of DNA damage is likely to have a lower effect then inhibiting Wee1 and Myt1 with Adavosertib or PD-166285. (F) Upon overexpression of Myt1 in the presence of UCN-01 or AZD6738 which leads to more inhibitory phosphorylation of Cdk1 on both T14 and Y15, the pool is reverted to more inactive Cdk1/cyclin B1 complexes even in the presence of these DNA damage checkpoint kinase inhibitors. Red arrows are Cdk1 activating pathways whereas black arrows are Cdk1 inactivating pathways. Solid line shows an active pathway, a dotted line an inhibited pathway. The p53 pathway (grey) is abrogated in most cancers. The meter schematically indicates the balance in active/inactive Cdk1 complexes in each scenario and the associated risk of premature entry into mitosis.
    Figure Legend Snippet: DNA damage and cell cycle checkpoint inhibitors and their effect on ectopic Cdk1 activation. (A) In an unperturbed cell, Cdk1 and cyclin B bind in late S-phase. Phosphorylation of Cdk1 on T14 and Y15 (depicted with red circles) by Myt1 and Wee1 during interphase inhibits Cdk1/cyclin B. Cdc25 removes these inhibitory phosphorylations and activates the Cdk1/cyclin B complexes allowing cells to enter mitosis. The high level of Wee1 activity in many cancer cells prevents their premature entry into mitosis and facilitates their survival in the presence of elevated genotoxic stress compared to normal cells. (B) Upon addition of Adavosertib (inhibition of Wee1) or PD166285 (inhibition of Wee1 and Myt1), the balance between inactive and active Cdk1/cyclin B1 complexes is shifted leading to more active Cdk1/cyclin B1 complexes. This increases the chance of premature entry into mitosis and cell death. (C) Overexpression of Myt1 in the presence of Adavosertib or PD166285 counters the drugs’ effect because phosphorylation of Cdk1 on T14 and Y15 by Myt1 increases inactive Cdk1/cyclin B complexes. This results in checkpoint arrest and rescue from cell death even in the presence of these cell cycle checkpoint kinase inhibitors. (D) Damaged DNA or replication stress can induce the activation of the DNA damage checkpoint, which downregulates Cdk1/cyclin B activity via Cdc25. ATR activates the effector kinase Chk1, which phosphorylates and inhibits the Cdc25 phosphatase family. In parallel, ATR can induce the stabilization and transcriptional activity of p53 leading to increased expression of the CDK inhibitor p21; however, this pathway is downregulated in many cancers (due to non-functional p53 status of many cancers) . (E) Upon addition of UCN-01 or AZD6738 (inhibition of Chk1 and ATR), Cdc25 inactivation in the presence of DNA damage is repressed and the balance between inactive and active Cdk1/cyclin B1 complexes is shifted towards more active Cdk1/cyclin B1 complexes. The cells are at elevated risk of premature mitotic entry. Restoration of Cdc25 activity in the presence of DNA damage is likely to have a lower effect then inhibiting Wee1 and Myt1 with Adavosertib or PD-166285. (F) Upon overexpression of Myt1 in the presence of UCN-01 or AZD6738 which leads to more inhibitory phosphorylation of Cdk1 on both T14 and Y15, the pool is reverted to more inactive Cdk1/cyclin B1 complexes even in the presence of these DNA damage checkpoint kinase inhibitors. Red arrows are Cdk1 activating pathways whereas black arrows are Cdk1 inactivating pathways. Solid line shows an active pathway, a dotted line an inhibited pathway. The p53 pathway (grey) is abrogated in most cancers. The meter schematically indicates the balance in active/inactive Cdk1 complexes in each scenario and the associated risk of premature entry into mitosis.

    Techniques Used: Activation Assay, Activity Assay, Inhibition, Over Expression, Expressing, Functional Assay

    phospho chk1 ser 345  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho chk1 ser 345
    Phospho Chk1 Ser 345, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phospho chk1 ser 345  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho chk1 ser 345
    ( A ) WT or E6AP knockout (KO) HeLa cells were treated with or without MASTL siRNA. The cells were incubated in 0.1 μM etoposide (ETO) for 18 hr, and released in fresh medium for recovery. Cells were harvested at the indicated time points (after the removal of ETO) for immunofluorescence (IF) using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified manually and shown. The mean values and standard deviations were calculated from three experiments. An unpaired two-tailed Student’s t test was used to determine the statistical significance (*p<0.05, **p<0.01, n>500 cell number/measurement). MASTL knockdown by siRNA was shown by immunoblotting in the panel E. ( B ) WT or E6AP KO HeLa cells with or without MASTL siRNA, as in panel A, were treated with 2 mM hydroxyurea (HU) for 18 hr. Cells were then released in fresh medium, and incubated as indicated, for recovery. The cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS), as described in Materials and methods. ( C ) WT or E6AP KO HeLa cells were treated with or without 0.5 μM doxorubicin (DOX) for 4 hr. Cells were then analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, phospho-SMC1 <t>Ser-957,</t> <t>phospho-CHK1</t> <t>Ser-345,</t> phospho-CHK2 Thr-68, γ-H2AX, and α-tubulin. ( D ) WT, E6AP KO, or E6AP KO with expression of HA-E6AP HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, and α-tubulin. ( E ) WT, E6AP KO, or E6AP KO with transfection of MASTL siRNA HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for MASTL, phospho-ATM/ATR substrates, and α-tubulin.
    Phospho Chk1 Ser 345, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery"

    Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

    Journal: eLife

    doi: 10.7554/eLife.86976

    ( A ) WT or E6AP knockout (KO) HeLa cells were treated with or without MASTL siRNA. The cells were incubated in 0.1 μM etoposide (ETO) for 18 hr, and released in fresh medium for recovery. Cells were harvested at the indicated time points (after the removal of ETO) for immunofluorescence (IF) using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified manually and shown. The mean values and standard deviations were calculated from three experiments. An unpaired two-tailed Student’s t test was used to determine the statistical significance (*p<0.05, **p<0.01, n>500 cell number/measurement). MASTL knockdown by siRNA was shown by immunoblotting in the panel E. ( B ) WT or E6AP KO HeLa cells with or without MASTL siRNA, as in panel A, were treated with 2 mM hydroxyurea (HU) for 18 hr. Cells were then released in fresh medium, and incubated as indicated, for recovery. The cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS), as described in Materials and methods. ( C ) WT or E6AP KO HeLa cells were treated with or without 0.5 μM doxorubicin (DOX) for 4 hr. Cells were then analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, phospho-CHK1 Ser-345, phospho-CHK2 Thr-68, γ-H2AX, and α-tubulin. ( D ) WT, E6AP KO, or E6AP KO with expression of HA-E6AP HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, and α-tubulin. ( E ) WT, E6AP KO, or E6AP KO with transfection of MASTL siRNA HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for MASTL, phospho-ATM/ATR substrates, and α-tubulin.
    Figure Legend Snippet: ( A ) WT or E6AP knockout (KO) HeLa cells were treated with or without MASTL siRNA. The cells were incubated in 0.1 μM etoposide (ETO) for 18 hr, and released in fresh medium for recovery. Cells were harvested at the indicated time points (after the removal of ETO) for immunofluorescence (IF) using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified manually and shown. The mean values and standard deviations were calculated from three experiments. An unpaired two-tailed Student’s t test was used to determine the statistical significance (*p<0.05, **p<0.01, n>500 cell number/measurement). MASTL knockdown by siRNA was shown by immunoblotting in the panel E. ( B ) WT or E6AP KO HeLa cells with or without MASTL siRNA, as in panel A, were treated with 2 mM hydroxyurea (HU) for 18 hr. Cells were then released in fresh medium, and incubated as indicated, for recovery. The cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS), as described in Materials and methods. ( C ) WT or E6AP KO HeLa cells were treated with or without 0.5 μM doxorubicin (DOX) for 4 hr. Cells were then analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, phospho-CHK1 Ser-345, phospho-CHK2 Thr-68, γ-H2AX, and α-tubulin. ( D ) WT, E6AP KO, or E6AP KO with expression of HA-E6AP HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, and α-tubulin. ( E ) WT, E6AP KO, or E6AP KO with transfection of MASTL siRNA HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for MASTL, phospho-ATM/ATR substrates, and α-tubulin.

    Techniques Used: Knock-Out, Incubation, Immunofluorescence, Activation Assay, Two Tailed Test, Western Blot, Fluorescence, FACS, Expressing, Transfection

    ( A ) HeLa cells expressing CFP-MASTL were treated without or with 10 mM hydroxyurea (HU) for 2 hr. CFP-MASTL immunoprecipitation (IP) was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, and β-actin. ( B ) HeLa cells expressing CFP-MASTL were treated without or with 10 mM HU and 4 mM caffeine, as indicated, for 2 hr. CFP-MASTL IP was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, phospho-CHK1 Ser-345, and α-tubulin.
    Figure Legend Snippet: ( A ) HeLa cells expressing CFP-MASTL were treated without or with 10 mM hydroxyurea (HU) for 2 hr. CFP-MASTL immunoprecipitation (IP) was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, and β-actin. ( B ) HeLa cells expressing CFP-MASTL were treated without or with 10 mM HU and 4 mM caffeine, as indicated, for 2 hr. CFP-MASTL IP was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, phospho-CHK1 Ser-345, and α-tubulin.

    Techniques Used: Expressing, Immunoprecipitation, Western Blot

    phospho chk1 ser 345  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho chk1 ser 345
    (A) WT or E6AP KO HeLa cells were treated with or without MASTL siRNA. The cells were incubated in 0.1 μM ETO for 18 hours, and released in fresh medium for recovery. Cells were harvested at the indicated time points (after the removal of ETO) for IF using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified manually and shown. The mean values and standard deviations were calculated from three experiments. An unpaired 2-tailed Student’s t test was used to determine the statistical significance (* p<0.05, ** p<0.01, n>500 cell number/measurement). MASTL knockdown by siRNA was shown by immunoblotting in the panel E. (B) WT or E6AP KO HeLa cells with or without MASTL siRNA, as in panel A, were treated with 2 mM HU for 18 hours. Cells were then released in fresh medium, and incubated as indicated, for recovery. The cell cycle progression was analyzed by Fluorescence-Activated Cell Sorting (FACS), as described in Materials and Methods. (C) WT or E6AP KO HeLa cells were treated with or without 0.5 μM DOX for 4 hours. Cells were then analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, phospho-SMC1 <t>Ser-957,</t> <t>phospho-CHK1</t> <t>Ser-345,</t> phospho-CHK2 Thr-68, γ-H2AX and α-tubulin. (D) WT, E6AP KO, or E6AP KO with expression of HA-E6AP HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates and α-tubulin. (E) WT, E6AP KO, or E6AP KO with transfection of MASTL siRNA HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates and α-tubulin.
    Phospho Chk1 Ser 345, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery"

    Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

    Journal: bioRxiv

    doi: 10.1101/2023.02.22.529521

    (A) WT or E6AP KO HeLa cells were treated with or without MASTL siRNA. The cells were incubated in 0.1 μM ETO for 18 hours, and released in fresh medium for recovery. Cells were harvested at the indicated time points (after the removal of ETO) for IF using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified manually and shown. The mean values and standard deviations were calculated from three experiments. An unpaired 2-tailed Student’s t test was used to determine the statistical significance (* p<0.05, ** p<0.01, n>500 cell number/measurement). MASTL knockdown by siRNA was shown by immunoblotting in the panel E. (B) WT or E6AP KO HeLa cells with or without MASTL siRNA, as in panel A, were treated with 2 mM HU for 18 hours. Cells were then released in fresh medium, and incubated as indicated, for recovery. The cell cycle progression was analyzed by Fluorescence-Activated Cell Sorting (FACS), as described in Materials and Methods. (C) WT or E6AP KO HeLa cells were treated with or without 0.5 μM DOX for 4 hours. Cells were then analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, phospho-CHK1 Ser-345, phospho-CHK2 Thr-68, γ-H2AX and α-tubulin. (D) WT, E6AP KO, or E6AP KO with expression of HA-E6AP HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates and α-tubulin. (E) WT, E6AP KO, or E6AP KO with transfection of MASTL siRNA HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates and α-tubulin.
    Figure Legend Snippet: (A) WT or E6AP KO HeLa cells were treated with or without MASTL siRNA. The cells were incubated in 0.1 μM ETO for 18 hours, and released in fresh medium for recovery. Cells were harvested at the indicated time points (after the removal of ETO) for IF using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified manually and shown. The mean values and standard deviations were calculated from three experiments. An unpaired 2-tailed Student’s t test was used to determine the statistical significance (* p<0.05, ** p<0.01, n>500 cell number/measurement). MASTL knockdown by siRNA was shown by immunoblotting in the panel E. (B) WT or E6AP KO HeLa cells with or without MASTL siRNA, as in panel A, were treated with 2 mM HU for 18 hours. Cells were then released in fresh medium, and incubated as indicated, for recovery. The cell cycle progression was analyzed by Fluorescence-Activated Cell Sorting (FACS), as described in Materials and Methods. (C) WT or E6AP KO HeLa cells were treated with or without 0.5 μM DOX for 4 hours. Cells were then analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, phospho-CHK1 Ser-345, phospho-CHK2 Thr-68, γ-H2AX and α-tubulin. (D) WT, E6AP KO, or E6AP KO with expression of HA-E6AP HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates and α-tubulin. (E) WT, E6AP KO, or E6AP KO with transfection of MASTL siRNA HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates and α-tubulin.

    Techniques Used: Incubation, Activation Assay, Knockdown, Western Blot, Fluorescence, FACS, Expressing, Transfection

    (A) HeLa cells expressing CFP-MASTL were treated without or with 10 mM HU for 2 hr. CFP-MASTL IP was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, and α-tubulin. (B) HeLa cells expressing CFP-MASTL were treated without or with 10 mM HU and 4 mM caffeine, as indicated, for 2 hr. CFP-MASTL IP was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, phospho-CHK1 Ser-345, and α-tubulin.
    Figure Legend Snippet: (A) HeLa cells expressing CFP-MASTL were treated without or with 10 mM HU for 2 hr. CFP-MASTL IP was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, and α-tubulin. (B) HeLa cells expressing CFP-MASTL were treated without or with 10 mM HU and 4 mM caffeine, as indicated, for 2 hr. CFP-MASTL IP was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, phospho-CHK1 Ser-345, and α-tubulin.

    Techniques Used: Expressing, Control, Western Blot

    phospho chk1 ser 345 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho chk1 ser 345 antibody
    Phospho Chk1 Ser 345 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phospho chk1 ser 345  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho chk1 ser 345
    (A) WT or E6AP KO HeLa cells were treated with or without MASTL siRNA. The cells were incubated in 0.1 μM ETO for 18 hours, and released in fresh medium for recovery. Cells were harvested at the indicated time points (after the removal of ETO) for IF using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified and shown. The mean values and standard deviations were calculated from three experiments. An unpaired 2-tailed Student’s t test was used to determine the statistical significance (* p<0.05, ** p<0.01). MASTL knockdown by siRNA was shown by immunoblotting in the panel E. (B) WT or E6AP KO HeLa cells with or without MASTL siRNA, as in panel A, were treated with 2 mM HU for 18 hours. Cells were then released in fresh medium, and incubated as indicated, for recovery. The cell cycle progression was analyzed by Fluorescence-Activated Cell Sorting (FACS), as described in Materials and Methods. (C) WT or E6AP KO HeLa cells were treated with or without 0.5 μM DOX for 4 hours. Cells were then analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, phospho-SMC1 <t>Ser-957,</t> <t>phospho-CHK1</t> <t>Ser-345,</t> phospho-CHK2 Thr-68, γ-H2AX and α-tubulin. (D) WT, E6AP KO, or E6AP KO with expression of HA-E6AP HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates and α-tubulin. (E) WT, E6AP KO, or E6AP KO with transfection of MASTL siRNA HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates and α-tubulin.
    Phospho Chk1 Ser 345, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery"

    Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

    Journal: bioRxiv

    doi: 10.1101/2023.02.22.529521

    (A) WT or E6AP KO HeLa cells were treated with or without MASTL siRNA. The cells were incubated in 0.1 μM ETO for 18 hours, and released in fresh medium for recovery. Cells were harvested at the indicated time points (after the removal of ETO) for IF using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified and shown. The mean values and standard deviations were calculated from three experiments. An unpaired 2-tailed Student’s t test was used to determine the statistical significance (* p<0.05, ** p<0.01). MASTL knockdown by siRNA was shown by immunoblotting in the panel E. (B) WT or E6AP KO HeLa cells with or without MASTL siRNA, as in panel A, were treated with 2 mM HU for 18 hours. Cells were then released in fresh medium, and incubated as indicated, for recovery. The cell cycle progression was analyzed by Fluorescence-Activated Cell Sorting (FACS), as described in Materials and Methods. (C) WT or E6AP KO HeLa cells were treated with or without 0.5 μM DOX for 4 hours. Cells were then analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, phospho-CHK1 Ser-345, phospho-CHK2 Thr-68, γ-H2AX and α-tubulin. (D) WT, E6AP KO, or E6AP KO with expression of HA-E6AP HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates and α-tubulin. (E) WT, E6AP KO, or E6AP KO with transfection of MASTL siRNA HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates and α-tubulin.
    Figure Legend Snippet: (A) WT or E6AP KO HeLa cells were treated with or without MASTL siRNA. The cells were incubated in 0.1 μM ETO for 18 hours, and released in fresh medium for recovery. Cells were harvested at the indicated time points (after the removal of ETO) for IF using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified and shown. The mean values and standard deviations were calculated from three experiments. An unpaired 2-tailed Student’s t test was used to determine the statistical significance (* p<0.05, ** p<0.01). MASTL knockdown by siRNA was shown by immunoblotting in the panel E. (B) WT or E6AP KO HeLa cells with or without MASTL siRNA, as in panel A, were treated with 2 mM HU for 18 hours. Cells were then released in fresh medium, and incubated as indicated, for recovery. The cell cycle progression was analyzed by Fluorescence-Activated Cell Sorting (FACS), as described in Materials and Methods. (C) WT or E6AP KO HeLa cells were treated with or without 0.5 μM DOX for 4 hours. Cells were then analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, phospho-CHK1 Ser-345, phospho-CHK2 Thr-68, γ-H2AX and α-tubulin. (D) WT, E6AP KO, or E6AP KO with expression of HA-E6AP HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates and α-tubulin. (E) WT, E6AP KO, or E6AP KO with transfection of MASTL siRNA HeLa cells were treated with or without 0.1 μM ETO, and analyzed by immunoblotting for E6AP, phospho-ATM/ATR substrates and α-tubulin.

    Techniques Used: Incubation, Activation Assay, Western Blot, Fluorescence, FACS, Expressing, Transfection

    (A) HeLa cells expressing CFP-MASTL were treated without or with 10 mM HU. CFP-MASTL IP was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, and α-tubulin. (B) HeLa cells expressing CFP-MASTL were treated without or with 10 mM HU and 4 mM caffeine, as indicated. CFP-MASTL IP was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, phospho-CHK1 Ser-345, and α-tubulin.
    Figure Legend Snippet: (A) HeLa cells expressing CFP-MASTL were treated without or with 10 mM HU. CFP-MASTL IP was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, and α-tubulin. (B) HeLa cells expressing CFP-MASTL were treated without or with 10 mM HU and 4 mM caffeine, as indicated. CFP-MASTL IP was performed using a GFP antibody. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for E6AP, MASTL, phospho-CHK1 Ser-345, and α-tubulin.

    Techniques Used: Expressing, Western Blot

    anti phosphorylated chk1 ser 345  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phosphorylated chk1 ser 345
    Anti Phosphorylated Chk1 Ser 345, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit monoclonal anti phospho chk1 ser 345 antibody 133d3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal anti phospho chk1 ser 345 antibody 133d3
    ( A ) Consensus motif for <t>Chk1</t> as suggested by O’Neill and co-workers . X, no particular amino acid preference; hy, hydrophobic amino acid; ba, basic amino acid. ( B ) Surrounding sequences of potential phosphorylation sites for Chk1 within the C-terminal domain of CK1δ, determined according to the published consensus sequence . ( C ) The wild-type GST-CK1δ fusion proteins FP1006, FP1022, and FP1183 were generated according to the positions of the predicted Chk1 phosphorylation sites within the C-terminal domain of rat CK1δ.
    Rabbit Monoclonal Anti Phospho Chk1 Ser 345 Antibody 133d3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "CK1δ Kinase Activity Is Modulated by Chk1-Mediated Phosphorylation"

    Article Title: CK1δ Kinase Activity Is Modulated by Chk1-Mediated Phosphorylation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068803

    ( A ) Consensus motif for Chk1 as suggested by O’Neill and co-workers . X, no particular amino acid preference; hy, hydrophobic amino acid; ba, basic amino acid. ( B ) Surrounding sequences of potential phosphorylation sites for Chk1 within the C-terminal domain of CK1δ, determined according to the published consensus sequence . ( C ) The wild-type GST-CK1δ fusion proteins FP1006, FP1022, and FP1183 were generated according to the positions of the predicted Chk1 phosphorylation sites within the C-terminal domain of rat CK1δ.
    Figure Legend Snippet: ( A ) Consensus motif for Chk1 as suggested by O’Neill and co-workers . X, no particular amino acid preference; hy, hydrophobic amino acid; ba, basic amino acid. ( B ) Surrounding sequences of potential phosphorylation sites for Chk1 within the C-terminal domain of CK1δ, determined according to the published consensus sequence . ( C ) The wild-type GST-CK1δ fusion proteins FP1006, FP1022, and FP1183 were generated according to the positions of the predicted Chk1 phosphorylation sites within the C-terminal domain of rat CK1δ.

    Techniques Used: Sequencing, Generated

    Chk1-mediated phosphorylation of three C-terminal CK1δ fusion protein sets containing either wild-type or mutant sequences encompassing amino acids 305–375 ( A ), 353–375 ( B ), and 375–428 ( C ) of the rat CK1δ sequence. The GST-CK1δ fusion proteins were phosphorylated by Chk1 in vitro and separated in SDS-PAGE. Substrate phosphorylation was quantified by Cherenkov counting. Results are shown as normalized bar graphs.
    Figure Legend Snippet: Chk1-mediated phosphorylation of three C-terminal CK1δ fusion protein sets containing either wild-type or mutant sequences encompassing amino acids 305–375 ( A ), 353–375 ( B ), and 375–428 ( C ) of the rat CK1δ sequence. The GST-CK1δ fusion proteins were phosphorylated by Chk1 in vitro and separated in SDS-PAGE. Substrate phosphorylation was quantified by Cherenkov counting. Results are shown as normalized bar graphs.

    Techniques Used: Mutagenesis, Sequencing, In Vitro, SDS Page

    Fusion proteins GST-CK1δ 305–375 (FP1006) and GST-CK1δ 305–375 S328A (FP1269) ( A ); GST-CK1δ 353–375 (FP1022) and GST-CK1δ 353–375 S370A (FP1021) ( B ); GST-CK1δ 375–428 (FP1183) and GST-CK1δ 375–428 T397A (FP1221) ( C ) were phosphorylated by Chk1 in vitro , processed and analyzed by two-dimensional phosphopeptide analyses as described in the Materials and Methods section. Arrow positions indicate identical phosphopeptide positions. Subsequent phosphoamino acid analysis of the indicated peptide from (A) is shown in panel ( D ). Mixed analyses confirm the identity of the arrow-marked peptides.
    Figure Legend Snippet: Fusion proteins GST-CK1δ 305–375 (FP1006) and GST-CK1δ 305–375 S328A (FP1269) ( A ); GST-CK1δ 353–375 (FP1022) and GST-CK1δ 353–375 S370A (FP1021) ( B ); GST-CK1δ 375–428 (FP1183) and GST-CK1δ 375–428 T397A (FP1221) ( C ) were phosphorylated by Chk1 in vitro , processed and analyzed by two-dimensional phosphopeptide analyses as described in the Materials and Methods section. Arrow positions indicate identical phosphopeptide positions. Subsequent phosphoamino acid analysis of the indicated peptide from (A) is shown in panel ( D ). Mixed analyses confirm the identity of the arrow-marked peptides.

    Techniques Used: In Vitro, Phosphoamino Acid Analysis

    ( A ) Alignment of the rat CK1δ sequence with the human CK1δ transcription variants (TV) 1 and 2. ( B ) GST-CK1δ 375–428 rat (FP1183), GST-CK1δ 375–415 TV1 (human, FP1341), and GST-CK1δ 375–409 TV2 (human, FP1343) were phosphorylated by Chk1 in vitro . The phosphorylated proteins were separated by SDS-PAGE and Chk1-mediated phosphorylation was quantified by Cherenkov counting of phosphorylated substrate bands. Results are shown as normalized bar graph. ( C ) GST-CK1δ 375–428 rat (FP1183), GST-CK1δ 375–415 TV1 (FP1341), and GST-CK1δ 375–409 TV2 (FP1343) were phosphorylated by Chk1 in vitro and processed for two-dimensional phosphopeptide analyses as described in the Materials and Methods section.
    Figure Legend Snippet: ( A ) Alignment of the rat CK1δ sequence with the human CK1δ transcription variants (TV) 1 and 2. ( B ) GST-CK1δ 375–428 rat (FP1183), GST-CK1δ 375–415 TV1 (human, FP1341), and GST-CK1δ 375–409 TV2 (human, FP1343) were phosphorylated by Chk1 in vitro . The phosphorylated proteins were separated by SDS-PAGE and Chk1-mediated phosphorylation was quantified by Cherenkov counting of phosphorylated substrate bands. Results are shown as normalized bar graph. ( C ) GST-CK1δ 375–428 rat (FP1183), GST-CK1δ 375–415 TV1 (FP1341), and GST-CK1δ 375–409 TV2 (FP1343) were phosphorylated by Chk1 in vitro and processed for two-dimensional phosphopeptide analyses as described in the Materials and Methods section.

    Techniques Used: Sequencing, In Vitro, SDS Page

    ( A ) The kinetic parameters K m and V max of GST-wt CK1δ (FP449) and generated phosphorylation-site mutants were determined by in vitro kinase assays using α-casein as substrate. Substrate phosphorylation was quantified by Cherenkov counting and data were fitted to the Michaelis-Menten equation. V max is expressed as pmol phosphate transferred per minute per mg of recombinant kinase. ( B ) GST-CK1δ was pre-incubated with activated Chk1 which was precipitated from hydroxyurea-treated HT1080 cells (Chk1(IP)) for 10 min. Subsequently, GST-β-catenin 1–181 was phosphorylated by GST-CK1δ alone or after pre-incubation with Chk1(IP) for additional 30 min. Data are presented as normalized bar graph.
    Figure Legend Snippet: ( A ) The kinetic parameters K m and V max of GST-wt CK1δ (FP449) and generated phosphorylation-site mutants were determined by in vitro kinase assays using α-casein as substrate. Substrate phosphorylation was quantified by Cherenkov counting and data were fitted to the Michaelis-Menten equation. V max is expressed as pmol phosphate transferred per minute per mg of recombinant kinase. ( B ) GST-CK1δ was pre-incubated with activated Chk1 which was precipitated from hydroxyurea-treated HT1080 cells (Chk1(IP)) for 10 min. Subsequently, GST-β-catenin 1–181 was phosphorylated by GST-CK1δ alone or after pre-incubation with Chk1(IP) for additional 30 min. Data are presented as normalized bar graph.

    Techniques Used: Generated, In Vitro, Recombinant, Incubation

    ( A ) Kinase assays were performed in the presence or absence of either 5 nM of compound 17 or 20 nM of compound 8 using GST-p53 1–64 (FP267) as substrate and GST-wt CK1δ or GST-CK1δ S328A, S370A, T397A as enzymes. * Observed effects are significant at p<0.05. ( B ) Kinase assays were performed in the presence or absence of either D4476 (300 nM), compound 17 (10 nM) or compound 8 (20 nM) using GST-p53 1–64 (FP267) as substrate and GST-wt CK1δ alone or in combination with Chk1 as enzymes. * Observed effects are significant at p<0.05.
    Figure Legend Snippet: ( A ) Kinase assays were performed in the presence or absence of either 5 nM of compound 17 or 20 nM of compound 8 using GST-p53 1–64 (FP267) as substrate and GST-wt CK1δ or GST-CK1δ S328A, S370A, T397A as enzymes. * Observed effects are significant at p<0.05. ( B ) Kinase assays were performed in the presence or absence of either D4476 (300 nM), compound 17 (10 nM) or compound 8 (20 nM) using GST-p53 1–64 (FP267) as substrate and GST-wt CK1δ alone or in combination with Chk1 as enzymes. * Observed effects are significant at p<0.05.

    Techniques Used:

    Chk1 was precipitated from 250 µg of extracts from untreated or hydroxyurea-treated (2.5 mM, 2 h) HT1080 cells using 2 µg of a Chk1-specific antibody. Immunoprecipitation (IP) of Chk1 and co-precipitation of CK1δ was detected by Western blot. Experiments using non-specific serum (control) or no antibody at all served as negative controls. Detection of β-actin in the lysate input served as loading control.
    Figure Legend Snippet: Chk1 was precipitated from 250 µg of extracts from untreated or hydroxyurea-treated (2.5 mM, 2 h) HT1080 cells using 2 µg of a Chk1-specific antibody. Immunoprecipitation (IP) of Chk1 and co-precipitation of CK1δ was detected by Western blot. Experiments using non-specific serum (control) or no antibody at all served as negative controls. Detection of β-actin in the lysate input served as loading control.

    Techniques Used: Immunoprecipitation, Western Blot

    Cellular Chk1 was activated by treating HT1080 cells with 2.5 mM hydroxyurea (HU) for the indicated periods of time. ( A ) Activation of Chk1 (indicated by phosphorylated Ser-345) and expression levels of Chk1 and CK1δ were determined by immunoblotting. Detection of β-actin served as loading control. ( B ) CK1 kinase activity in fractionated extracts from HT1080 cells before and after treatment with 2.5 mM HU for 2, 4, 6 and 8 hours, respectively, was determined using GST-p53 1–64 (FP267) as substrate. The detected kinase activity was normalized towards the untreated control. ( C ) Presence of CK1δ in the kinase peak fractions shown in (B) was confirmed by use of the CK1δ-specific inhibitor compound 17 at 50 nM . ( D ) HT1080 cells were treated with 2.5 mM HU and/or the Chk1-specific inhibitor SB-218078 for 2 h. CK1 kinase activity in fractionated extracts was determined using GST-p53 1–64 (FP267) as substrate. The detected kinase activity was normalized towards the untreated control.
    Figure Legend Snippet: Cellular Chk1 was activated by treating HT1080 cells with 2.5 mM hydroxyurea (HU) for the indicated periods of time. ( A ) Activation of Chk1 (indicated by phosphorylated Ser-345) and expression levels of Chk1 and CK1δ were determined by immunoblotting. Detection of β-actin served as loading control. ( B ) CK1 kinase activity in fractionated extracts from HT1080 cells before and after treatment with 2.5 mM HU for 2, 4, 6 and 8 hours, respectively, was determined using GST-p53 1–64 (FP267) as substrate. The detected kinase activity was normalized towards the untreated control. ( C ) Presence of CK1δ in the kinase peak fractions shown in (B) was confirmed by use of the CK1δ-specific inhibitor compound 17 at 50 nM . ( D ) HT1080 cells were treated with 2.5 mM HU and/or the Chk1-specific inhibitor SB-218078 for 2 h. CK1 kinase activity in fractionated extracts was determined using GST-p53 1–64 (FP267) as substrate. The detected kinase activity was normalized towards the untreated control.

    Techniques Used: Activation Assay, Expressing, Western Blot, Activity Assay

    s 345 p chk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc s 345 p chk1
    UV-C induced checkpoint activation in PrimPol −/− cells is partially resolved by inhibition of <t>Chk1</t> or p38. (A) Chk1 phosphorylation was analyzed by western blotting of whole cell lysates at increasing recovery times (2-24 hrs) after 4 J/m 2 UV-C damage. (B) The affect of UCN-01 on cell cycle progression was measured by counting the presence of p-H3 positive mitotic cells. Cells were pre-treated with 100 nM UCN-01 for approximately 2 hrs before irradiation with 0 or 4 J/m 2 UV-C, cells were allowed to recover for 0 or 16 hr before the addition of nocadozole to block mitotic exit for 4 hrs. (C) Mitotic segregation was analyzed by staining with DAPI and α-tubulin 16 hrs after cells were damaged with 4 J/m 2 in this case cells were pre-treated and then maintained in 100 nM UCN-01 prior to damage. (D) Effect of p38 on cell cycle progression was measured by counting the percentage of p-H3 positive mitotic cells 4 hrs after incubation with nocodazole. Cells were first pre-treated with 2.5 μM SB203580 for 2 hrs followed by irradiation with 4 J/m 2 UV-C, and a 16 hr recovery period. (E) The ability of checkpoint inhibitors to release cells from G2 arrest was measured by allowing cells to recover after 0 or 4 J/m 2 UV-C for 16 hrs, followed by addition of 100 nM UCN-01 or 2.5 µM SB203580 and 0.5 µM nocodazole for 4 hrs. Mitotic entry was then assessed by p-H3 staining. For all experiments n ≥ 3 independent experiments, error bars represent standard deviation.
    S 345 P Chk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "PrimPol-deficient cells exhibit a pronounced G2 checkpoint response following UV damage"

    Article Title: PrimPol-deficient cells exhibit a pronounced G2 checkpoint response following UV damage

    Journal: Cell Cycle

    doi: 10.1080/15384101.2015.1128597

    UV-C induced checkpoint activation in PrimPol −/− cells is partially resolved by inhibition of Chk1 or p38. (A) Chk1 phosphorylation was analyzed by western blotting of whole cell lysates at increasing recovery times (2-24 hrs) after 4 J/m 2 UV-C damage. (B) The affect of UCN-01 on cell cycle progression was measured by counting the presence of p-H3 positive mitotic cells. Cells were pre-treated with 100 nM UCN-01 for approximately 2 hrs before irradiation with 0 or 4 J/m 2 UV-C, cells were allowed to recover for 0 or 16 hr before the addition of nocadozole to block mitotic exit for 4 hrs. (C) Mitotic segregation was analyzed by staining with DAPI and α-tubulin 16 hrs after cells were damaged with 4 J/m 2 in this case cells were pre-treated and then maintained in 100 nM UCN-01 prior to damage. (D) Effect of p38 on cell cycle progression was measured by counting the percentage of p-H3 positive mitotic cells 4 hrs after incubation with nocodazole. Cells were first pre-treated with 2.5 μM SB203580 for 2 hrs followed by irradiation with 4 J/m 2 UV-C, and a 16 hr recovery period. (E) The ability of checkpoint inhibitors to release cells from G2 arrest was measured by allowing cells to recover after 0 or 4 J/m 2 UV-C for 16 hrs, followed by addition of 100 nM UCN-01 or 2.5 µM SB203580 and 0.5 µM nocodazole for 4 hrs. Mitotic entry was then assessed by p-H3 staining. For all experiments n ≥ 3 independent experiments, error bars represent standard deviation.
    Figure Legend Snippet: UV-C induced checkpoint activation in PrimPol −/− cells is partially resolved by inhibition of Chk1 or p38. (A) Chk1 phosphorylation was analyzed by western blotting of whole cell lysates at increasing recovery times (2-24 hrs) after 4 J/m 2 UV-C damage. (B) The affect of UCN-01 on cell cycle progression was measured by counting the presence of p-H3 positive mitotic cells. Cells were pre-treated with 100 nM UCN-01 for approximately 2 hrs before irradiation with 0 or 4 J/m 2 UV-C, cells were allowed to recover for 0 or 16 hr before the addition of nocadozole to block mitotic exit for 4 hrs. (C) Mitotic segregation was analyzed by staining with DAPI and α-tubulin 16 hrs after cells were damaged with 4 J/m 2 in this case cells were pre-treated and then maintained in 100 nM UCN-01 prior to damage. (D) Effect of p38 on cell cycle progression was measured by counting the percentage of p-H3 positive mitotic cells 4 hrs after incubation with nocodazole. Cells were first pre-treated with 2.5 μM SB203580 for 2 hrs followed by irradiation with 4 J/m 2 UV-C, and a 16 hr recovery period. (E) The ability of checkpoint inhibitors to release cells from G2 arrest was measured by allowing cells to recover after 0 or 4 J/m 2 UV-C for 16 hrs, followed by addition of 100 nM UCN-01 or 2.5 µM SB203580 and 0.5 µM nocodazole for 4 hrs. Mitotic entry was then assessed by p-H3 staining. For all experiments n ≥ 3 independent experiments, error bars represent standard deviation.

    Techniques Used: Activation Assay, Inhibition, Western Blot, Irradiation, Blocking Assay, Staining, Incubation, Standard Deviation

    PrimPol −/− cells are more resistant to Chk1 inhibition than WT cells. (A) Cell viability was measured using Cell Titer Blue, 48 hrs after 4 J/m 2 UV-C damage, where cells were maintained in 100 nM UCN-01. (B) Colony formation was analyzed in the presence of 2 mM caffeine after increasing doses of UV-C damage in comparison with survival in the absence of the inhibitor. (C) Cell Titer Blue was used to measure cell viability 48 hrs after 4 J/m 2 UV-C damage, where cells were pre-treated and maintained in 2.5 µM of SB203580, p38 inhibitor. Error bars represent standard deviation of independent repeats and significance was analysed using a students T-test (*p < 0.05, **p < 0.01).
    Figure Legend Snippet: PrimPol −/− cells are more resistant to Chk1 inhibition than WT cells. (A) Cell viability was measured using Cell Titer Blue, 48 hrs after 4 J/m 2 UV-C damage, where cells were maintained in 100 nM UCN-01. (B) Colony formation was analyzed in the presence of 2 mM caffeine after increasing doses of UV-C damage in comparison with survival in the absence of the inhibitor. (C) Cell Titer Blue was used to measure cell viability 48 hrs after 4 J/m 2 UV-C damage, where cells were pre-treated and maintained in 2.5 µM of SB203580, p38 inhibitor. Error bars represent standard deviation of independent repeats and significance was analysed using a students T-test (*p < 0.05, **p < 0.01).

    Techniques Used: Inhibition, Standard Deviation

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    Cell Signaling Technology Inc rabbit monoclonal anti phospho chk1 ser 345
    HCT116, HT-29 and SiHa cancer cells were chronically adapted at pH 6.5 or maintained at pH 7.4 ( A , C , D ) or native HCT116 cells were acutely exposed to acidic pH e ( B ) or treated with 5-FU at the indicated doses to be compared with cancer cells adapted at pH 6.5 ( E , F ). ( A – C ) Representative immunoblots of total and phosphorylated ATM, ATR, <t>CHK1,</t> CHK2. Actin or tubulin was used as loading control, as indicated. ( D ) Bar graph showing the proportion of tetraploid cells. ( E , F ) Representative immunoblots of total and phosphorylated ATM, ATR, CHK1, CHK2. Actin was used as loading control, as indicated. ( G – J ) Cell viability assays in HCT116 ( G , I ) and HT-29 cancer cells ( H , J ) cultured at pH 7.4 or 6.5, and treated with the indicated doses of ATMi AZD0156 ( G , H ) or ATRi elimusertib ( I , J ) for 72 h. Data information: ( A – J ) data represent n = 3 independent biological replicates. ( D , G – J ) Bar graphs represent means ± SD with three biological replicates ( D ) or six technical replicates ( G – J ), and significance was determined using two-way ANOVA with Tukey’s multiple-comparison analysis (ns non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). .
    Rabbit Monoclonal Anti Phospho Chk1 Ser 345, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti phospho serine 345 chk1
    Myt1 overexpression inhibits mitotic entry by reducing Cdk1 activity in cells treated with checkpoint kinase inhibitors (A) HeLa cells were seeded onto coverslips in the presence or absence of tetracycline (2 µM) for 24 h. Cells were synchronized in G1-S phase by single thymidine block. Following synchronization cells were released into media containing kinase inhibitors or DMSO for 4 h. Cells were stained for DNA and PH3 and analyzed by immunofluorescence microscopy. The percentage of cells positive for PH3 is shown normalized to DMSO/Tet-. Error bars represent SEM. Experiments were repeated three times. Statistical significance was determined by 2-way ANOVA. ***, p < 0.001; **, p = 0.001. (B) Cell lysates were prepared from tetracycline inducible Myt1 expressing HeLa cells. Cells were treated or not with 2 µM of tetracycline 48 h prior to any drug treatment/control: DMSO, Adavosertib (500 nM), PD166285 (500 nM), UCN-01 (1000 nM), and AZD6738 (1000 nM) for 4 h. Cdk1 activity in lysates from cells was assessed in vitro by incubation with GST-PP1Cα (a Cdk1 substrate). Total pT320-PP1Cα peptide and GST levels were determined by immunoblot. (C) Bar graphs show the quantitation of the absolute Cdk1 activity in cells treated with various checkpoint kinase inhibitors. Error bars represent SD. Statistical analysis was done using a 2-way ANOVA. ****, p < 0.0001**, p = 0.0029. (D) Lysates were also evaluated for the levels of pY15-Cdk1, Cdk1, <t>pS345-Chk1,</t> Chk1, overexpressed Myt1 and Tubulin. Quantitation shows the average pS345-Chk1 and pY15-Cdk1 levels relative to their total proteins and normalized to DMSO with no tetracycline addition. Kinase assay was repeated 5 times or more. Western blotting and the immunofluorescence experiments were repeated three times.
    Rabbit Anti Phospho Serine 345 Chk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho chk1 ser 345
    Myt1 overexpression inhibits mitotic entry by reducing Cdk1 activity in cells treated with checkpoint kinase inhibitors (A) HeLa cells were seeded onto coverslips in the presence or absence of tetracycline (2 µM) for 24 h. Cells were synchronized in G1-S phase by single thymidine block. Following synchronization cells were released into media containing kinase inhibitors or DMSO for 4 h. Cells were stained for DNA and PH3 and analyzed by immunofluorescence microscopy. The percentage of cells positive for PH3 is shown normalized to DMSO/Tet-. Error bars represent SEM. Experiments were repeated three times. Statistical significance was determined by 2-way ANOVA. ***, p < 0.001; **, p = 0.001. (B) Cell lysates were prepared from tetracycline inducible Myt1 expressing HeLa cells. Cells were treated or not with 2 µM of tetracycline 48 h prior to any drug treatment/control: DMSO, Adavosertib (500 nM), PD166285 (500 nM), UCN-01 (1000 nM), and AZD6738 (1000 nM) for 4 h. Cdk1 activity in lysates from cells was assessed in vitro by incubation with GST-PP1Cα (a Cdk1 substrate). Total pT320-PP1Cα peptide and GST levels were determined by immunoblot. (C) Bar graphs show the quantitation of the absolute Cdk1 activity in cells treated with various checkpoint kinase inhibitors. Error bars represent SD. Statistical analysis was done using a 2-way ANOVA. ****, p < 0.0001**, p = 0.0029. (D) Lysates were also evaluated for the levels of pY15-Cdk1, Cdk1, <t>pS345-Chk1,</t> Chk1, overexpressed Myt1 and Tubulin. Quantitation shows the average pS345-Chk1 and pY15-Cdk1 levels relative to their total proteins and normalized to DMSO with no tetracycline addition. Kinase assay was repeated 5 times or more. Western blotting and the immunofluorescence experiments were repeated three times.
    Phospho Chk1 Ser 345, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho chk1 ser 345 antibody
    Myt1 overexpression inhibits mitotic entry by reducing Cdk1 activity in cells treated with checkpoint kinase inhibitors (A) HeLa cells were seeded onto coverslips in the presence or absence of tetracycline (2 µM) for 24 h. Cells were synchronized in G1-S phase by single thymidine block. Following synchronization cells were released into media containing kinase inhibitors or DMSO for 4 h. Cells were stained for DNA and PH3 and analyzed by immunofluorescence microscopy. The percentage of cells positive for PH3 is shown normalized to DMSO/Tet-. Error bars represent SEM. Experiments were repeated three times. Statistical significance was determined by 2-way ANOVA. ***, p < 0.001; **, p = 0.001. (B) Cell lysates were prepared from tetracycline inducible Myt1 expressing HeLa cells. Cells were treated or not with 2 µM of tetracycline 48 h prior to any drug treatment/control: DMSO, Adavosertib (500 nM), PD166285 (500 nM), UCN-01 (1000 nM), and AZD6738 (1000 nM) for 4 h. Cdk1 activity in lysates from cells was assessed in vitro by incubation with GST-PP1Cα (a Cdk1 substrate). Total pT320-PP1Cα peptide and GST levels were determined by immunoblot. (C) Bar graphs show the quantitation of the absolute Cdk1 activity in cells treated with various checkpoint kinase inhibitors. Error bars represent SD. Statistical analysis was done using a 2-way ANOVA. ****, p < 0.0001**, p = 0.0029. (D) Lysates were also evaluated for the levels of pY15-Cdk1, Cdk1, <t>pS345-Chk1,</t> Chk1, overexpressed Myt1 and Tubulin. Quantitation shows the average pS345-Chk1 and pY15-Cdk1 levels relative to their total proteins and normalized to DMSO with no tetracycline addition. Kinase assay was repeated 5 times or more. Western blotting and the immunofluorescence experiments were repeated three times.
    Phospho Chk1 Ser 345 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phosphorylated chk1 ser 345
    Myt1 overexpression inhibits mitotic entry by reducing Cdk1 activity in cells treated with checkpoint kinase inhibitors (A) HeLa cells were seeded onto coverslips in the presence or absence of tetracycline (2 µM) for 24 h. Cells were synchronized in G1-S phase by single thymidine block. Following synchronization cells were released into media containing kinase inhibitors or DMSO for 4 h. Cells were stained for DNA and PH3 and analyzed by immunofluorescence microscopy. The percentage of cells positive for PH3 is shown normalized to DMSO/Tet-. Error bars represent SEM. Experiments were repeated three times. Statistical significance was determined by 2-way ANOVA. ***, p < 0.001; **, p = 0.001. (B) Cell lysates were prepared from tetracycline inducible Myt1 expressing HeLa cells. Cells were treated or not with 2 µM of tetracycline 48 h prior to any drug treatment/control: DMSO, Adavosertib (500 nM), PD166285 (500 nM), UCN-01 (1000 nM), and AZD6738 (1000 nM) for 4 h. Cdk1 activity in lysates from cells was assessed in vitro by incubation with GST-PP1Cα (a Cdk1 substrate). Total pT320-PP1Cα peptide and GST levels were determined by immunoblot. (C) Bar graphs show the quantitation of the absolute Cdk1 activity in cells treated with various checkpoint kinase inhibitors. Error bars represent SD. Statistical analysis was done using a 2-way ANOVA. ****, p < 0.0001**, p = 0.0029. (D) Lysates were also evaluated for the levels of pY15-Cdk1, Cdk1, <t>pS345-Chk1,</t> Chk1, overexpressed Myt1 and Tubulin. Quantitation shows the average pS345-Chk1 and pY15-Cdk1 levels relative to their total proteins and normalized to DMSO with no tetracycline addition. Kinase assay was repeated 5 times or more. Western blotting and the immunofluorescence experiments were repeated three times.
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    Cell Signaling Technology Inc rabbit monoclonal anti phospho chk1 ser 345 antibody 133d3
    ( A ) Consensus motif for <t>Chk1</t> as suggested by O’Neill and co-workers . X, no particular amino acid preference; hy, hydrophobic amino acid; ba, basic amino acid. ( B ) Surrounding sequences of potential phosphorylation sites for Chk1 within the C-terminal domain of CK1δ, determined according to the published consensus sequence . ( C ) The wild-type GST-CK1δ fusion proteins FP1006, FP1022, and FP1183 were generated according to the positions of the predicted Chk1 phosphorylation sites within the C-terminal domain of rat CK1δ.
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    Cell Signaling Technology Inc s 345 p chk1
    UV-C induced checkpoint activation in PrimPol −/− cells is partially resolved by inhibition of <t>Chk1</t> or p38. (A) Chk1 phosphorylation was analyzed by western blotting of whole cell lysates at increasing recovery times (2-24 hrs) after 4 J/m 2 UV-C damage. (B) The affect of UCN-01 on cell cycle progression was measured by counting the presence of p-H3 positive mitotic cells. Cells were pre-treated with 100 nM UCN-01 for approximately 2 hrs before irradiation with 0 or 4 J/m 2 UV-C, cells were allowed to recover for 0 or 16 hr before the addition of nocadozole to block mitotic exit for 4 hrs. (C) Mitotic segregation was analyzed by staining with DAPI and α-tubulin 16 hrs after cells were damaged with 4 J/m 2 in this case cells were pre-treated and then maintained in 100 nM UCN-01 prior to damage. (D) Effect of p38 on cell cycle progression was measured by counting the percentage of p-H3 positive mitotic cells 4 hrs after incubation with nocodazole. Cells were first pre-treated with 2.5 μM SB203580 for 2 hrs followed by irradiation with 4 J/m 2 UV-C, and a 16 hr recovery period. (E) The ability of checkpoint inhibitors to release cells from G2 arrest was measured by allowing cells to recover after 0 or 4 J/m 2 UV-C for 16 hrs, followed by addition of 100 nM UCN-01 or 2.5 µM SB203580 and 0.5 µM nocodazole for 4 hrs. Mitotic entry was then assessed by p-H3 staining. For all experiments n ≥ 3 independent experiments, error bars represent standard deviation.
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    HCT116, HT-29 and SiHa cancer cells were chronically adapted at pH 6.5 or maintained at pH 7.4 ( A , C , D ) or native HCT116 cells were acutely exposed to acidic pH e ( B ) or treated with 5-FU at the indicated doses to be compared with cancer cells adapted at pH 6.5 ( E , F ). ( A – C ) Representative immunoblots of total and phosphorylated ATM, ATR, CHK1, CHK2. Actin or tubulin was used as loading control, as indicated. ( D ) Bar graph showing the proportion of tetraploid cells. ( E , F ) Representative immunoblots of total and phosphorylated ATM, ATR, CHK1, CHK2. Actin was used as loading control, as indicated. ( G – J ) Cell viability assays in HCT116 ( G , I ) and HT-29 cancer cells ( H , J ) cultured at pH 7.4 or 6.5, and treated with the indicated doses of ATMi AZD0156 ( G , H ) or ATRi elimusertib ( I , J ) for 72 h. Data information: ( A – J ) data represent n = 3 independent biological replicates. ( D , G – J ) Bar graphs represent means ± SD with three biological replicates ( D ) or six technical replicates ( G – J ), and significance was determined using two-way ANOVA with Tukey’s multiple-comparison analysis (ns non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). .

    Journal: EMBO Reports

    Article Title: Tumor acidosis-induced DNA damage response and tetraploidy enhance sensitivity to ATM and ATR inhibitors

    doi: 10.1038/s44319-024-00089-7

    Figure Lengend Snippet: HCT116, HT-29 and SiHa cancer cells were chronically adapted at pH 6.5 or maintained at pH 7.4 ( A , C , D ) or native HCT116 cells were acutely exposed to acidic pH e ( B ) or treated with 5-FU at the indicated doses to be compared with cancer cells adapted at pH 6.5 ( E , F ). ( A – C ) Representative immunoblots of total and phosphorylated ATM, ATR, CHK1, CHK2. Actin or tubulin was used as loading control, as indicated. ( D ) Bar graph showing the proportion of tetraploid cells. ( E , F ) Representative immunoblots of total and phosphorylated ATM, ATR, CHK1, CHK2. Actin was used as loading control, as indicated. ( G – J ) Cell viability assays in HCT116 ( G , I ) and HT-29 cancer cells ( H , J ) cultured at pH 7.4 or 6.5, and treated with the indicated doses of ATMi AZD0156 ( G , H ) or ATRi elimusertib ( I , J ) for 72 h. Data information: ( A – J ) data represent n = 3 independent biological replicates. ( D , G – J ) Bar graphs represent means ± SD with three biological replicates ( D ) or six technical replicates ( G – J ), and significance was determined using two-way ANOVA with Tukey’s multiple-comparison analysis (ns non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). .

    Article Snippet: Rabbit monoclonal anti-phospho-CHK1 (Ser-345) (clone 133D3) , Cell Signaling Technology , Cat# 2348; RRID: AB_331212.

    Techniques: Western Blot, Cell Culture, Comparison

    Reagents and tools.

    Journal: EMBO Reports

    Article Title: Tumor acidosis-induced DNA damage response and tetraploidy enhance sensitivity to ATM and ATR inhibitors

    doi: 10.1038/s44319-024-00089-7

    Figure Lengend Snippet: Reagents and tools.

    Article Snippet: Rabbit monoclonal anti-phospho-CHK1 (Ser-345) (clone 133D3) , Cell Signaling Technology , Cat# 2348; RRID: AB_331212.

    Techniques: Membrane, Modification, Protease Inhibitor, Software, Bicinchoninic Acid Protein Assay, Viability Assay

    Myt1 overexpression inhibits mitotic entry by reducing Cdk1 activity in cells treated with checkpoint kinase inhibitors (A) HeLa cells were seeded onto coverslips in the presence or absence of tetracycline (2 µM) for 24 h. Cells were synchronized in G1-S phase by single thymidine block. Following synchronization cells were released into media containing kinase inhibitors or DMSO for 4 h. Cells were stained for DNA and PH3 and analyzed by immunofluorescence microscopy. The percentage of cells positive for PH3 is shown normalized to DMSO/Tet-. Error bars represent SEM. Experiments were repeated three times. Statistical significance was determined by 2-way ANOVA. ***, p < 0.001; **, p = 0.001. (B) Cell lysates were prepared from tetracycline inducible Myt1 expressing HeLa cells. Cells were treated or not with 2 µM of tetracycline 48 h prior to any drug treatment/control: DMSO, Adavosertib (500 nM), PD166285 (500 nM), UCN-01 (1000 nM), and AZD6738 (1000 nM) for 4 h. Cdk1 activity in lysates from cells was assessed in vitro by incubation with GST-PP1Cα (a Cdk1 substrate). Total pT320-PP1Cα peptide and GST levels were determined by immunoblot. (C) Bar graphs show the quantitation of the absolute Cdk1 activity in cells treated with various checkpoint kinase inhibitors. Error bars represent SD. Statistical analysis was done using a 2-way ANOVA. ****, p < 0.0001**, p = 0.0029. (D) Lysates were also evaluated for the levels of pY15-Cdk1, Cdk1, pS345-Chk1, Chk1, overexpressed Myt1 and Tubulin. Quantitation shows the average pS345-Chk1 and pY15-Cdk1 levels relative to their total proteins and normalized to DMSO with no tetracycline addition. Kinase assay was repeated 5 times or more. Western blotting and the immunofluorescence experiments were repeated three times.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors

    doi: 10.3389/fcell.2023.1270542

    Figure Lengend Snippet: Myt1 overexpression inhibits mitotic entry by reducing Cdk1 activity in cells treated with checkpoint kinase inhibitors (A) HeLa cells were seeded onto coverslips in the presence or absence of tetracycline (2 µM) for 24 h. Cells were synchronized in G1-S phase by single thymidine block. Following synchronization cells were released into media containing kinase inhibitors or DMSO for 4 h. Cells were stained for DNA and PH3 and analyzed by immunofluorescence microscopy. The percentage of cells positive for PH3 is shown normalized to DMSO/Tet-. Error bars represent SEM. Experiments were repeated three times. Statistical significance was determined by 2-way ANOVA. ***, p < 0.001; **, p = 0.001. (B) Cell lysates were prepared from tetracycline inducible Myt1 expressing HeLa cells. Cells were treated or not with 2 µM of tetracycline 48 h prior to any drug treatment/control: DMSO, Adavosertib (500 nM), PD166285 (500 nM), UCN-01 (1000 nM), and AZD6738 (1000 nM) for 4 h. Cdk1 activity in lysates from cells was assessed in vitro by incubation with GST-PP1Cα (a Cdk1 substrate). Total pT320-PP1Cα peptide and GST levels were determined by immunoblot. (C) Bar graphs show the quantitation of the absolute Cdk1 activity in cells treated with various checkpoint kinase inhibitors. Error bars represent SD. Statistical analysis was done using a 2-way ANOVA. ****, p < 0.0001**, p = 0.0029. (D) Lysates were also evaluated for the levels of pY15-Cdk1, Cdk1, pS345-Chk1, Chk1, overexpressed Myt1 and Tubulin. Quantitation shows the average pS345-Chk1 and pY15-Cdk1 levels relative to their total proteins and normalized to DMSO with no tetracycline addition. Kinase assay was repeated 5 times or more. Western blotting and the immunofluorescence experiments were repeated three times.

    Article Snippet: Proteins were stained using rabbit anti-phospho-tyrosine 15-Cdk1 (Cell Signaling Technology; 9,111; 1:1,000 dilution), rabbit anti-phospho-threonine 14-Cdk1 (as described in ); 1:1,000 dilution), mouse anti-Cdc2 p34 (Santa Cruz Biotechnology; sc-54; 1:1,000 dilution), rabbit anti-Wee1 (Cell Signaling Technology; 4,936; 1:1,000 dilution), anti-pT320-PP1Cα antibody (Abcam; ab62334; 1:30,000 dilution), anti-GST antibody (Rockland; 600-401-200; 1:2,000 dilution), rabbit anti-phospho-serine 345-Chk1 (Cell Signaling Technology; 2,348; 1:1,000 dilution), mouse anti-Chk1 (Cell Signaling Technology; 2,360; 1:5,000), anti-tubulin antibody (Sigma; T5168; 1:10,000 dilution), anti-Myt1 antibody (Cell Signaling Technology; 4,282; 1:500 dilution), and anti-GFP IR800-Conjugated antibody (Rockland; 600-132-215; 1:1,000 dilution).

    Techniques: Over Expression, Activity Assay, Blocking Assay, Staining, Immunofluorescence, Microscopy, Expressing, In Vitro, Incubation, Western Blot, Quantitation Assay, Kinase Assay

    DNA damage and cell cycle checkpoint inhibitors and their effect on ectopic Cdk1 activation. (A) In an unperturbed cell, Cdk1 and cyclin B bind in late S-phase. Phosphorylation of Cdk1 on T14 and Y15 (depicted with red circles) by Myt1 and Wee1 during interphase inhibits Cdk1/cyclin B. Cdc25 removes these inhibitory phosphorylations and activates the Cdk1/cyclin B complexes allowing cells to enter mitosis. The high level of Wee1 activity in many cancer cells prevents their premature entry into mitosis and facilitates their survival in the presence of elevated genotoxic stress compared to normal cells. (B) Upon addition of Adavosertib (inhibition of Wee1) or PD166285 (inhibition of Wee1 and Myt1), the balance between inactive and active Cdk1/cyclin B1 complexes is shifted leading to more active Cdk1/cyclin B1 complexes. This increases the chance of premature entry into mitosis and cell death. (C) Overexpression of Myt1 in the presence of Adavosertib or PD166285 counters the drugs’ effect because phosphorylation of Cdk1 on T14 and Y15 by Myt1 increases inactive Cdk1/cyclin B complexes. This results in checkpoint arrest and rescue from cell death even in the presence of these cell cycle checkpoint kinase inhibitors. (D) Damaged DNA or replication stress can induce the activation of the DNA damage checkpoint, which downregulates Cdk1/cyclin B activity via Cdc25. ATR activates the effector kinase Chk1, which phosphorylates and inhibits the Cdc25 phosphatase family. In parallel, ATR can induce the stabilization and transcriptional activity of p53 leading to increased expression of the CDK inhibitor p21; however, this pathway is downregulated in many cancers (due to non-functional p53 status of many cancers) . (E) Upon addition of UCN-01 or AZD6738 (inhibition of Chk1 and ATR), Cdc25 inactivation in the presence of DNA damage is repressed and the balance between inactive and active Cdk1/cyclin B1 complexes is shifted towards more active Cdk1/cyclin B1 complexes. The cells are at elevated risk of premature mitotic entry. Restoration of Cdc25 activity in the presence of DNA damage is likely to have a lower effect then inhibiting Wee1 and Myt1 with Adavosertib or PD-166285. (F) Upon overexpression of Myt1 in the presence of UCN-01 or AZD6738 which leads to more inhibitory phosphorylation of Cdk1 on both T14 and Y15, the pool is reverted to more inactive Cdk1/cyclin B1 complexes even in the presence of these DNA damage checkpoint kinase inhibitors. Red arrows are Cdk1 activating pathways whereas black arrows are Cdk1 inactivating pathways. Solid line shows an active pathway, a dotted line an inhibited pathway. The p53 pathway (grey) is abrogated in most cancers. The meter schematically indicates the balance in active/inactive Cdk1 complexes in each scenario and the associated risk of premature entry into mitosis.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors

    doi: 10.3389/fcell.2023.1270542

    Figure Lengend Snippet: DNA damage and cell cycle checkpoint inhibitors and their effect on ectopic Cdk1 activation. (A) In an unperturbed cell, Cdk1 and cyclin B bind in late S-phase. Phosphorylation of Cdk1 on T14 and Y15 (depicted with red circles) by Myt1 and Wee1 during interphase inhibits Cdk1/cyclin B. Cdc25 removes these inhibitory phosphorylations and activates the Cdk1/cyclin B complexes allowing cells to enter mitosis. The high level of Wee1 activity in many cancer cells prevents their premature entry into mitosis and facilitates their survival in the presence of elevated genotoxic stress compared to normal cells. (B) Upon addition of Adavosertib (inhibition of Wee1) or PD166285 (inhibition of Wee1 and Myt1), the balance between inactive and active Cdk1/cyclin B1 complexes is shifted leading to more active Cdk1/cyclin B1 complexes. This increases the chance of premature entry into mitosis and cell death. (C) Overexpression of Myt1 in the presence of Adavosertib or PD166285 counters the drugs’ effect because phosphorylation of Cdk1 on T14 and Y15 by Myt1 increases inactive Cdk1/cyclin B complexes. This results in checkpoint arrest and rescue from cell death even in the presence of these cell cycle checkpoint kinase inhibitors. (D) Damaged DNA or replication stress can induce the activation of the DNA damage checkpoint, which downregulates Cdk1/cyclin B activity via Cdc25. ATR activates the effector kinase Chk1, which phosphorylates and inhibits the Cdc25 phosphatase family. In parallel, ATR can induce the stabilization and transcriptional activity of p53 leading to increased expression of the CDK inhibitor p21; however, this pathway is downregulated in many cancers (due to non-functional p53 status of many cancers) . (E) Upon addition of UCN-01 or AZD6738 (inhibition of Chk1 and ATR), Cdc25 inactivation in the presence of DNA damage is repressed and the balance between inactive and active Cdk1/cyclin B1 complexes is shifted towards more active Cdk1/cyclin B1 complexes. The cells are at elevated risk of premature mitotic entry. Restoration of Cdc25 activity in the presence of DNA damage is likely to have a lower effect then inhibiting Wee1 and Myt1 with Adavosertib or PD-166285. (F) Upon overexpression of Myt1 in the presence of UCN-01 or AZD6738 which leads to more inhibitory phosphorylation of Cdk1 on both T14 and Y15, the pool is reverted to more inactive Cdk1/cyclin B1 complexes even in the presence of these DNA damage checkpoint kinase inhibitors. Red arrows are Cdk1 activating pathways whereas black arrows are Cdk1 inactivating pathways. Solid line shows an active pathway, a dotted line an inhibited pathway. The p53 pathway (grey) is abrogated in most cancers. The meter schematically indicates the balance in active/inactive Cdk1 complexes in each scenario and the associated risk of premature entry into mitosis.

    Article Snippet: Proteins were stained using rabbit anti-phospho-tyrosine 15-Cdk1 (Cell Signaling Technology; 9,111; 1:1,000 dilution), rabbit anti-phospho-threonine 14-Cdk1 (as described in ); 1:1,000 dilution), mouse anti-Cdc2 p34 (Santa Cruz Biotechnology; sc-54; 1:1,000 dilution), rabbit anti-Wee1 (Cell Signaling Technology; 4,936; 1:1,000 dilution), anti-pT320-PP1Cα antibody (Abcam; ab62334; 1:30,000 dilution), anti-GST antibody (Rockland; 600-401-200; 1:2,000 dilution), rabbit anti-phospho-serine 345-Chk1 (Cell Signaling Technology; 2,348; 1:1,000 dilution), mouse anti-Chk1 (Cell Signaling Technology; 2,360; 1:5,000), anti-tubulin antibody (Sigma; T5168; 1:10,000 dilution), anti-Myt1 antibody (Cell Signaling Technology; 4,282; 1:500 dilution), and anti-GFP IR800-Conjugated antibody (Rockland; 600-132-215; 1:1,000 dilution).

    Techniques: Activation Assay, Activity Assay, Inhibition, Over Expression, Expressing, Functional Assay

    ( A ) Consensus motif for Chk1 as suggested by O’Neill and co-workers . X, no particular amino acid preference; hy, hydrophobic amino acid; ba, basic amino acid. ( B ) Surrounding sequences of potential phosphorylation sites for Chk1 within the C-terminal domain of CK1δ, determined according to the published consensus sequence . ( C ) The wild-type GST-CK1δ fusion proteins FP1006, FP1022, and FP1183 were generated according to the positions of the predicted Chk1 phosphorylation sites within the C-terminal domain of rat CK1δ.

    Journal: PLoS ONE

    Article Title: CK1δ Kinase Activity Is Modulated by Chk1-Mediated Phosphorylation

    doi: 10.1371/journal.pone.0068803

    Figure Lengend Snippet: ( A ) Consensus motif for Chk1 as suggested by O’Neill and co-workers . X, no particular amino acid preference; hy, hydrophobic amino acid; ba, basic amino acid. ( B ) Surrounding sequences of potential phosphorylation sites for Chk1 within the C-terminal domain of CK1δ, determined according to the published consensus sequence . ( C ) The wild-type GST-CK1δ fusion proteins FP1006, FP1022, and FP1183 were generated according to the positions of the predicted Chk1 phosphorylation sites within the C-terminal domain of rat CK1δ.

    Article Snippet: Phosphorylated Chk1 (pChk1 S345 ) was detected using the rabbit monoclonal anti-phospho-Chk1 (Ser-345) antibody 133D3 (1∶500; 2348, Cell Signaling Technology, USA).

    Techniques: Sequencing, Generated

    Chk1-mediated phosphorylation of three C-terminal CK1δ fusion protein sets containing either wild-type or mutant sequences encompassing amino acids 305–375 ( A ), 353–375 ( B ), and 375–428 ( C ) of the rat CK1δ sequence. The GST-CK1δ fusion proteins were phosphorylated by Chk1 in vitro and separated in SDS-PAGE. Substrate phosphorylation was quantified by Cherenkov counting. Results are shown as normalized bar graphs.

    Journal: PLoS ONE

    Article Title: CK1δ Kinase Activity Is Modulated by Chk1-Mediated Phosphorylation

    doi: 10.1371/journal.pone.0068803

    Figure Lengend Snippet: Chk1-mediated phosphorylation of three C-terminal CK1δ fusion protein sets containing either wild-type or mutant sequences encompassing amino acids 305–375 ( A ), 353–375 ( B ), and 375–428 ( C ) of the rat CK1δ sequence. The GST-CK1δ fusion proteins were phosphorylated by Chk1 in vitro and separated in SDS-PAGE. Substrate phosphorylation was quantified by Cherenkov counting. Results are shown as normalized bar graphs.

    Article Snippet: Phosphorylated Chk1 (pChk1 S345 ) was detected using the rabbit monoclonal anti-phospho-Chk1 (Ser-345) antibody 133D3 (1∶500; 2348, Cell Signaling Technology, USA).

    Techniques: Mutagenesis, Sequencing, In Vitro, SDS Page

    Fusion proteins GST-CK1δ 305–375 (FP1006) and GST-CK1δ 305–375 S328A (FP1269) ( A ); GST-CK1δ 353–375 (FP1022) and GST-CK1δ 353–375 S370A (FP1021) ( B ); GST-CK1δ 375–428 (FP1183) and GST-CK1δ 375–428 T397A (FP1221) ( C ) were phosphorylated by Chk1 in vitro , processed and analyzed by two-dimensional phosphopeptide analyses as described in the Materials and Methods section. Arrow positions indicate identical phosphopeptide positions. Subsequent phosphoamino acid analysis of the indicated peptide from (A) is shown in panel ( D ). Mixed analyses confirm the identity of the arrow-marked peptides.

    Journal: PLoS ONE

    Article Title: CK1δ Kinase Activity Is Modulated by Chk1-Mediated Phosphorylation

    doi: 10.1371/journal.pone.0068803

    Figure Lengend Snippet: Fusion proteins GST-CK1δ 305–375 (FP1006) and GST-CK1δ 305–375 S328A (FP1269) ( A ); GST-CK1δ 353–375 (FP1022) and GST-CK1δ 353–375 S370A (FP1021) ( B ); GST-CK1δ 375–428 (FP1183) and GST-CK1δ 375–428 T397A (FP1221) ( C ) were phosphorylated by Chk1 in vitro , processed and analyzed by two-dimensional phosphopeptide analyses as described in the Materials and Methods section. Arrow positions indicate identical phosphopeptide positions. Subsequent phosphoamino acid analysis of the indicated peptide from (A) is shown in panel ( D ). Mixed analyses confirm the identity of the arrow-marked peptides.

    Article Snippet: Phosphorylated Chk1 (pChk1 S345 ) was detected using the rabbit monoclonal anti-phospho-Chk1 (Ser-345) antibody 133D3 (1∶500; 2348, Cell Signaling Technology, USA).

    Techniques: In Vitro, Phosphoamino Acid Analysis

    ( A ) Alignment of the rat CK1δ sequence with the human CK1δ transcription variants (TV) 1 and 2. ( B ) GST-CK1δ 375–428 rat (FP1183), GST-CK1δ 375–415 TV1 (human, FP1341), and GST-CK1δ 375–409 TV2 (human, FP1343) were phosphorylated by Chk1 in vitro . The phosphorylated proteins were separated by SDS-PAGE and Chk1-mediated phosphorylation was quantified by Cherenkov counting of phosphorylated substrate bands. Results are shown as normalized bar graph. ( C ) GST-CK1δ 375–428 rat (FP1183), GST-CK1δ 375–415 TV1 (FP1341), and GST-CK1δ 375–409 TV2 (FP1343) were phosphorylated by Chk1 in vitro and processed for two-dimensional phosphopeptide analyses as described in the Materials and Methods section.

    Journal: PLoS ONE

    Article Title: CK1δ Kinase Activity Is Modulated by Chk1-Mediated Phosphorylation

    doi: 10.1371/journal.pone.0068803

    Figure Lengend Snippet: ( A ) Alignment of the rat CK1δ sequence with the human CK1δ transcription variants (TV) 1 and 2. ( B ) GST-CK1δ 375–428 rat (FP1183), GST-CK1δ 375–415 TV1 (human, FP1341), and GST-CK1δ 375–409 TV2 (human, FP1343) were phosphorylated by Chk1 in vitro . The phosphorylated proteins were separated by SDS-PAGE and Chk1-mediated phosphorylation was quantified by Cherenkov counting of phosphorylated substrate bands. Results are shown as normalized bar graph. ( C ) GST-CK1δ 375–428 rat (FP1183), GST-CK1δ 375–415 TV1 (FP1341), and GST-CK1δ 375–409 TV2 (FP1343) were phosphorylated by Chk1 in vitro and processed for two-dimensional phosphopeptide analyses as described in the Materials and Methods section.

    Article Snippet: Phosphorylated Chk1 (pChk1 S345 ) was detected using the rabbit monoclonal anti-phospho-Chk1 (Ser-345) antibody 133D3 (1∶500; 2348, Cell Signaling Technology, USA).

    Techniques: Sequencing, In Vitro, SDS Page

    ( A ) The kinetic parameters K m and V max of GST-wt CK1δ (FP449) and generated phosphorylation-site mutants were determined by in vitro kinase assays using α-casein as substrate. Substrate phosphorylation was quantified by Cherenkov counting and data were fitted to the Michaelis-Menten equation. V max is expressed as pmol phosphate transferred per minute per mg of recombinant kinase. ( B ) GST-CK1δ was pre-incubated with activated Chk1 which was precipitated from hydroxyurea-treated HT1080 cells (Chk1(IP)) for 10 min. Subsequently, GST-β-catenin 1–181 was phosphorylated by GST-CK1δ alone or after pre-incubation with Chk1(IP) for additional 30 min. Data are presented as normalized bar graph.

    Journal: PLoS ONE

    Article Title: CK1δ Kinase Activity Is Modulated by Chk1-Mediated Phosphorylation

    doi: 10.1371/journal.pone.0068803

    Figure Lengend Snippet: ( A ) The kinetic parameters K m and V max of GST-wt CK1δ (FP449) and generated phosphorylation-site mutants were determined by in vitro kinase assays using α-casein as substrate. Substrate phosphorylation was quantified by Cherenkov counting and data were fitted to the Michaelis-Menten equation. V max is expressed as pmol phosphate transferred per minute per mg of recombinant kinase. ( B ) GST-CK1δ was pre-incubated with activated Chk1 which was precipitated from hydroxyurea-treated HT1080 cells (Chk1(IP)) for 10 min. Subsequently, GST-β-catenin 1–181 was phosphorylated by GST-CK1δ alone or after pre-incubation with Chk1(IP) for additional 30 min. Data are presented as normalized bar graph.

    Article Snippet: Phosphorylated Chk1 (pChk1 S345 ) was detected using the rabbit monoclonal anti-phospho-Chk1 (Ser-345) antibody 133D3 (1∶500; 2348, Cell Signaling Technology, USA).

    Techniques: Generated, In Vitro, Recombinant, Incubation

    ( A ) Kinase assays were performed in the presence or absence of either 5 nM of compound 17 or 20 nM of compound 8 using GST-p53 1–64 (FP267) as substrate and GST-wt CK1δ or GST-CK1δ S328A, S370A, T397A as enzymes. * Observed effects are significant at p<0.05. ( B ) Kinase assays were performed in the presence or absence of either D4476 (300 nM), compound 17 (10 nM) or compound 8 (20 nM) using GST-p53 1–64 (FP267) as substrate and GST-wt CK1δ alone or in combination with Chk1 as enzymes. * Observed effects are significant at p<0.05.

    Journal: PLoS ONE

    Article Title: CK1δ Kinase Activity Is Modulated by Chk1-Mediated Phosphorylation

    doi: 10.1371/journal.pone.0068803

    Figure Lengend Snippet: ( A ) Kinase assays were performed in the presence or absence of either 5 nM of compound 17 or 20 nM of compound 8 using GST-p53 1–64 (FP267) as substrate and GST-wt CK1δ or GST-CK1δ S328A, S370A, T397A as enzymes. * Observed effects are significant at p<0.05. ( B ) Kinase assays were performed in the presence or absence of either D4476 (300 nM), compound 17 (10 nM) or compound 8 (20 nM) using GST-p53 1–64 (FP267) as substrate and GST-wt CK1δ alone or in combination with Chk1 as enzymes. * Observed effects are significant at p<0.05.

    Article Snippet: Phosphorylated Chk1 (pChk1 S345 ) was detected using the rabbit monoclonal anti-phospho-Chk1 (Ser-345) antibody 133D3 (1∶500; 2348, Cell Signaling Technology, USA).

    Techniques:

    Chk1 was precipitated from 250 µg of extracts from untreated or hydroxyurea-treated (2.5 mM, 2 h) HT1080 cells using 2 µg of a Chk1-specific antibody. Immunoprecipitation (IP) of Chk1 and co-precipitation of CK1δ was detected by Western blot. Experiments using non-specific serum (control) or no antibody at all served as negative controls. Detection of β-actin in the lysate input served as loading control.

    Journal: PLoS ONE

    Article Title: CK1δ Kinase Activity Is Modulated by Chk1-Mediated Phosphorylation

    doi: 10.1371/journal.pone.0068803

    Figure Lengend Snippet: Chk1 was precipitated from 250 µg of extracts from untreated or hydroxyurea-treated (2.5 mM, 2 h) HT1080 cells using 2 µg of a Chk1-specific antibody. Immunoprecipitation (IP) of Chk1 and co-precipitation of CK1δ was detected by Western blot. Experiments using non-specific serum (control) or no antibody at all served as negative controls. Detection of β-actin in the lysate input served as loading control.

    Article Snippet: Phosphorylated Chk1 (pChk1 S345 ) was detected using the rabbit monoclonal anti-phospho-Chk1 (Ser-345) antibody 133D3 (1∶500; 2348, Cell Signaling Technology, USA).

    Techniques: Immunoprecipitation, Western Blot

    Cellular Chk1 was activated by treating HT1080 cells with 2.5 mM hydroxyurea (HU) for the indicated periods of time. ( A ) Activation of Chk1 (indicated by phosphorylated Ser-345) and expression levels of Chk1 and CK1δ were determined by immunoblotting. Detection of β-actin served as loading control. ( B ) CK1 kinase activity in fractionated extracts from HT1080 cells before and after treatment with 2.5 mM HU for 2, 4, 6 and 8 hours, respectively, was determined using GST-p53 1–64 (FP267) as substrate. The detected kinase activity was normalized towards the untreated control. ( C ) Presence of CK1δ in the kinase peak fractions shown in (B) was confirmed by use of the CK1δ-specific inhibitor compound 17 at 50 nM . ( D ) HT1080 cells were treated with 2.5 mM HU and/or the Chk1-specific inhibitor SB-218078 for 2 h. CK1 kinase activity in fractionated extracts was determined using GST-p53 1–64 (FP267) as substrate. The detected kinase activity was normalized towards the untreated control.

    Journal: PLoS ONE

    Article Title: CK1δ Kinase Activity Is Modulated by Chk1-Mediated Phosphorylation

    doi: 10.1371/journal.pone.0068803

    Figure Lengend Snippet: Cellular Chk1 was activated by treating HT1080 cells with 2.5 mM hydroxyurea (HU) for the indicated periods of time. ( A ) Activation of Chk1 (indicated by phosphorylated Ser-345) and expression levels of Chk1 and CK1δ were determined by immunoblotting. Detection of β-actin served as loading control. ( B ) CK1 kinase activity in fractionated extracts from HT1080 cells before and after treatment with 2.5 mM HU for 2, 4, 6 and 8 hours, respectively, was determined using GST-p53 1–64 (FP267) as substrate. The detected kinase activity was normalized towards the untreated control. ( C ) Presence of CK1δ in the kinase peak fractions shown in (B) was confirmed by use of the CK1δ-specific inhibitor compound 17 at 50 nM . ( D ) HT1080 cells were treated with 2.5 mM HU and/or the Chk1-specific inhibitor SB-218078 for 2 h. CK1 kinase activity in fractionated extracts was determined using GST-p53 1–64 (FP267) as substrate. The detected kinase activity was normalized towards the untreated control.

    Article Snippet: Phosphorylated Chk1 (pChk1 S345 ) was detected using the rabbit monoclonal anti-phospho-Chk1 (Ser-345) antibody 133D3 (1∶500; 2348, Cell Signaling Technology, USA).

    Techniques: Activation Assay, Expressing, Western Blot, Activity Assay

    UV-C induced checkpoint activation in PrimPol −/− cells is partially resolved by inhibition of Chk1 or p38. (A) Chk1 phosphorylation was analyzed by western blotting of whole cell lysates at increasing recovery times (2-24 hrs) after 4 J/m 2 UV-C damage. (B) The affect of UCN-01 on cell cycle progression was measured by counting the presence of p-H3 positive mitotic cells. Cells were pre-treated with 100 nM UCN-01 for approximately 2 hrs before irradiation with 0 or 4 J/m 2 UV-C, cells were allowed to recover for 0 or 16 hr before the addition of nocadozole to block mitotic exit for 4 hrs. (C) Mitotic segregation was analyzed by staining with DAPI and α-tubulin 16 hrs after cells were damaged with 4 J/m 2 in this case cells were pre-treated and then maintained in 100 nM UCN-01 prior to damage. (D) Effect of p38 on cell cycle progression was measured by counting the percentage of p-H3 positive mitotic cells 4 hrs after incubation with nocodazole. Cells were first pre-treated with 2.5 μM SB203580 for 2 hrs followed by irradiation with 4 J/m 2 UV-C, and a 16 hr recovery period. (E) The ability of checkpoint inhibitors to release cells from G2 arrest was measured by allowing cells to recover after 0 or 4 J/m 2 UV-C for 16 hrs, followed by addition of 100 nM UCN-01 or 2.5 µM SB203580 and 0.5 µM nocodazole for 4 hrs. Mitotic entry was then assessed by p-H3 staining. For all experiments n ≥ 3 independent experiments, error bars represent standard deviation.

    Journal: Cell Cycle

    Article Title: PrimPol-deficient cells exhibit a pronounced G2 checkpoint response following UV damage

    doi: 10.1080/15384101.2015.1128597

    Figure Lengend Snippet: UV-C induced checkpoint activation in PrimPol −/− cells is partially resolved by inhibition of Chk1 or p38. (A) Chk1 phosphorylation was analyzed by western blotting of whole cell lysates at increasing recovery times (2-24 hrs) after 4 J/m 2 UV-C damage. (B) The affect of UCN-01 on cell cycle progression was measured by counting the presence of p-H3 positive mitotic cells. Cells were pre-treated with 100 nM UCN-01 for approximately 2 hrs before irradiation with 0 or 4 J/m 2 UV-C, cells were allowed to recover for 0 or 16 hr before the addition of nocadozole to block mitotic exit for 4 hrs. (C) Mitotic segregation was analyzed by staining with DAPI and α-tubulin 16 hrs after cells were damaged with 4 J/m 2 in this case cells were pre-treated and then maintained in 100 nM UCN-01 prior to damage. (D) Effect of p38 on cell cycle progression was measured by counting the percentage of p-H3 positive mitotic cells 4 hrs after incubation with nocodazole. Cells were first pre-treated with 2.5 μM SB203580 for 2 hrs followed by irradiation with 4 J/m 2 UV-C, and a 16 hr recovery period. (E) The ability of checkpoint inhibitors to release cells from G2 arrest was measured by allowing cells to recover after 0 or 4 J/m 2 UV-C for 16 hrs, followed by addition of 100 nM UCN-01 or 2.5 µM SB203580 and 0.5 µM nocodazole for 4 hrs. Mitotic entry was then assessed by p-H3 staining. For all experiments n ≥ 3 independent experiments, error bars represent standard deviation.

    Article Snippet: Proteins of interest were detected by antibodies to S 345 P-Chk1 (Cell Signaling), α-tubulin (Sigma), Chk1 (Santa Cruz) and Hrp labeled secondary (Abcam and Dakko).

    Techniques: Activation Assay, Inhibition, Western Blot, Irradiation, Blocking Assay, Staining, Incubation, Standard Deviation

    PrimPol −/− cells are more resistant to Chk1 inhibition than WT cells. (A) Cell viability was measured using Cell Titer Blue, 48 hrs after 4 J/m 2 UV-C damage, where cells were maintained in 100 nM UCN-01. (B) Colony formation was analyzed in the presence of 2 mM caffeine after increasing doses of UV-C damage in comparison with survival in the absence of the inhibitor. (C) Cell Titer Blue was used to measure cell viability 48 hrs after 4 J/m 2 UV-C damage, where cells were pre-treated and maintained in 2.5 µM of SB203580, p38 inhibitor. Error bars represent standard deviation of independent repeats and significance was analysed using a students T-test (*p < 0.05, **p < 0.01).

    Journal: Cell Cycle

    Article Title: PrimPol-deficient cells exhibit a pronounced G2 checkpoint response following UV damage

    doi: 10.1080/15384101.2015.1128597

    Figure Lengend Snippet: PrimPol −/− cells are more resistant to Chk1 inhibition than WT cells. (A) Cell viability was measured using Cell Titer Blue, 48 hrs after 4 J/m 2 UV-C damage, where cells were maintained in 100 nM UCN-01. (B) Colony formation was analyzed in the presence of 2 mM caffeine after increasing doses of UV-C damage in comparison with survival in the absence of the inhibitor. (C) Cell Titer Blue was used to measure cell viability 48 hrs after 4 J/m 2 UV-C damage, where cells were pre-treated and maintained in 2.5 µM of SB203580, p38 inhibitor. Error bars represent standard deviation of independent repeats and significance was analysed using a students T-test (*p < 0.05, **p < 0.01).

    Article Snippet: Proteins of interest were detected by antibodies to S 345 P-Chk1 (Cell Signaling), α-tubulin (Sigma), Chk1 (Santa Cruz) and Hrp labeled secondary (Abcam and Dakko).

    Techniques: Inhibition, Standard Deviation