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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    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

    phospho chk1 ser 345  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho chk1 ser 345
    siRNA screen for genes that promote proliferation of MDA-MB-231 cells identify <t>CHK1,</t> RRM1 and RRM2 as top-hits . ( A ) Effects on proliferation by gene knockdown with the custom siRNA library. Data are shown as a z-score distribution from the mean. ( B ) Percent change (from non-targeting siRNA (NTS) control) in proliferation of cells due to knock-down of expression by individual siRNA oligos for the genes noted. Q-RT-PCR determination of reduced RNA ( C ) and protein ( D ) expression for CHK1, RRMI, and RRM2 for individual siRNA oligos. (E) Percent growth of cells due to expression of Qiagen CHK1-siRNA. Gene ( F ) and protein ( G ) expression for CHK1 is suppressed in cells that are transfected with individual Qiagen CHK1-siRNAs. Graphs shown are representative of 3 repeated experiments.
    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 "Cross-species genomic and functional analyses identify a combination therapy using a CHK1 inhibitor and a ribonucleotide reductase inhibitor to treat triple-negative breast cancer"

    Article Title: Cross-species genomic and functional analyses identify a combination therapy using a CHK1 inhibitor and a ribonucleotide reductase inhibitor to treat triple-negative breast cancer

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr3230

    siRNA screen for genes that promote proliferation of MDA-MB-231 cells identify CHK1, RRM1 and RRM2 as top-hits . ( A ) Effects on proliferation by gene knockdown with the custom siRNA library. Data are shown as a z-score distribution from the mean. ( B ) Percent change (from non-targeting siRNA (NTS) control) in proliferation of cells due to knock-down of expression by individual siRNA oligos for the genes noted. Q-RT-PCR determination of reduced RNA ( C ) and protein ( D ) expression for CHK1, RRMI, and RRM2 for individual siRNA oligos. (E) Percent growth of cells due to expression of Qiagen CHK1-siRNA. Gene ( F ) and protein ( G ) expression for CHK1 is suppressed in cells that are transfected with individual Qiagen CHK1-siRNAs. Graphs shown are representative of 3 repeated experiments.
    Figure Legend Snippet: siRNA screen for genes that promote proliferation of MDA-MB-231 cells identify CHK1, RRM1 and RRM2 as top-hits . ( A ) Effects on proliferation by gene knockdown with the custom siRNA library. Data are shown as a z-score distribution from the mean. ( B ) Percent change (from non-targeting siRNA (NTS) control) in proliferation of cells due to knock-down of expression by individual siRNA oligos for the genes noted. Q-RT-PCR determination of reduced RNA ( C ) and protein ( D ) expression for CHK1, RRMI, and RRM2 for individual siRNA oligos. (E) Percent growth of cells due to expression of Qiagen CHK1-siRNA. Gene ( F ) and protein ( G ) expression for CHK1 is suppressed in cells that are transfected with individual Qiagen CHK1-siRNAs. Graphs shown are representative of 3 repeated experiments.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection

    Combination therapy with CHK1 inhibitors and gemcitabine inhibits proliferation in TNBC cells . MDA-MB-231 ( A, C ) and M6 ( B, D ) cells were treated with agents on day 0 and proliferation was measured on days indicated. MDA-MB-231 cells (gemcitabine 10 nM; UCN-01 150 nM), (gemcitabine10 nM; AZD 7762 200 nM). M6 cells (gemcitabine 4 nM; UCN-01 20 nM) (gemcitabine 4 nM; AZD 7762 30 nM). Results in B and D are from one experiment thus Gem treatment and vehicle are the same in both panels. P -value based upon change from vehicle treatment (letters only) and from single agent to combination treatment (line and letter) (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).
    Figure Legend Snippet: Combination therapy with CHK1 inhibitors and gemcitabine inhibits proliferation in TNBC cells . MDA-MB-231 ( A, C ) and M6 ( B, D ) cells were treated with agents on day 0 and proliferation was measured on days indicated. MDA-MB-231 cells (gemcitabine 10 nM; UCN-01 150 nM), (gemcitabine10 nM; AZD 7762 200 nM). M6 cells (gemcitabine 4 nM; UCN-01 20 nM) (gemcitabine 4 nM; AZD 7762 30 nM). Results in B and D are from one experiment thus Gem treatment and vehicle are the same in both panels. P -value based upon change from vehicle treatment (letters only) and from single agent to combination treatment (line and letter) (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).

    Techniques Used:

    Combination therapy with UCN-01 and gemcitabine induces DNA damage and apoptosis in TNBC cells . ( A ) Protein samples were collected 24 hours after drug treatment to detect changes in DNA damage (gamma-H2AX), checkpoint activation (phos-CHK1 andtTotal CHK1) and cell cycle progression (Cyclin A) by immunoblot analysis. ( B ) Cell cycle changes were assessed by BrdU-labeling and propidium iodide staining 24 hour after drug treatment. ( C ) Percentage of cells at 48 hours in early apoptosis (Annexin V + /7-AAD - ) and late apoptosis (Annexin V+/7-AAD+) with representative data ( D ). MDA-MB-231 cells (gemcitabine 10 nM; UCN-01 150 nM), M6 cells (gemcitabine 4 nM; UCN-01 20 nM). P -value based upon change from vehicle treatment (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).
    Figure Legend Snippet: Combination therapy with UCN-01 and gemcitabine induces DNA damage and apoptosis in TNBC cells . ( A ) Protein samples were collected 24 hours after drug treatment to detect changes in DNA damage (gamma-H2AX), checkpoint activation (phos-CHK1 andtTotal CHK1) and cell cycle progression (Cyclin A) by immunoblot analysis. ( B ) Cell cycle changes were assessed by BrdU-labeling and propidium iodide staining 24 hour after drug treatment. ( C ) Percentage of cells at 48 hours in early apoptosis (Annexin V + /7-AAD - ) and late apoptosis (Annexin V+/7-AAD+) with representative data ( D ). MDA-MB-231 cells (gemcitabine 10 nM; UCN-01 150 nM), M6 cells (gemcitabine 4 nM; UCN-01 20 nM). P -value based upon change from vehicle treatment (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).

    Techniques Used: Activation Assay, Western Blot, Labeling, Staining

    Combination therapy with CHK1 inhibitors and gemcitabine inhibits proliferation in TNBC cells . BT-549 ( A ), SUM 159 ( B) and HCC 1187 (C ) cells were treated with agents on day 0 and proliferation was measured on days indicated. BT-549 cells (gemcitabine 10 nM; UCN-01 100 nM; AZD 7762 150 nM). SUM 159 (gemcitabine 4 nM; UCN-01 80 nM; AZD 7762 300 nM). HCC 1187 (gemcitabine 10 nM; UCN-01 150 nM; AZD 7762 200 nM). P -value based upon change from vehicle treatment (letters only) and from single agent to combination treatment (line and letter; day three only) (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).
    Figure Legend Snippet: Combination therapy with CHK1 inhibitors and gemcitabine inhibits proliferation in TNBC cells . BT-549 ( A ), SUM 159 ( B) and HCC 1187 (C ) cells were treated with agents on day 0 and proliferation was measured on days indicated. BT-549 cells (gemcitabine 10 nM; UCN-01 100 nM; AZD 7762 150 nM). SUM 159 (gemcitabine 4 nM; UCN-01 80 nM; AZD 7762 300 nM). HCC 1187 (gemcitabine 10 nM; UCN-01 150 nM; AZD 7762 200 nM). P -value based upon change from vehicle treatment (letters only) and from single agent to combination treatment (line and letter; day three only) (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).

    Techniques Used:

    In vivo response of TNBC tumors to agents that target CHK1, RRM1 and RRM2 . ( A ) SCID mice with MDA-MB-231 tumor xenografts were treated seven days after implantation with vehicle, 5 mg/kg gemcitabine, 6 mg/kg UCN-01, or a combination of both on a Q4Dx3 schedule. Gemcitabine was delivered first by IP injection and UCN-01 was delivered 24 hours later by IV injection on a Q6Hx2. Note: Inset graph highlights days 7 to 21. During this period, mice treated with the combination therapy had slower growing tumors than those treated only with gemcitabine. ( B ) SCID mice with C3(1)Tag tumor transplants were dosed 12 days after transplantation with 20 mg/kg gemcitabine, 4.5 mg/kg UCN-01, or the drug combination on a Q4Dx3 schedule. Gemcitabine was delivered first and UCN-01 was delivered eight hours later on a Q6Hx2 schedule.
    Figure Legend Snippet: In vivo response of TNBC tumors to agents that target CHK1, RRM1 and RRM2 . ( A ) SCID mice with MDA-MB-231 tumor xenografts were treated seven days after implantation with vehicle, 5 mg/kg gemcitabine, 6 mg/kg UCN-01, or a combination of both on a Q4Dx3 schedule. Gemcitabine was delivered first by IP injection and UCN-01 was delivered 24 hours later by IV injection on a Q6Hx2. Note: Inset graph highlights days 7 to 21. During this period, mice treated with the combination therapy had slower growing tumors than those treated only with gemcitabine. ( B ) SCID mice with C3(1)Tag tumor transplants were dosed 12 days after transplantation with 20 mg/kg gemcitabine, 4.5 mg/kg UCN-01, or the drug combination on a Q4Dx3 schedule. Gemcitabine was delivered first and UCN-01 was delivered eight hours later on a Q6Hx2 schedule.

    Techniques Used: In Vivo, Injection, IV Injection, Transplantation Assay

    rabbit monoclonal anti phospho chk1 ser 345 133d3 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal anti phospho chk1 ser 345 133d3 antibody
    Vpr induces cell cycle G2/M arrest through activation of <t>Chk1</t> via <t>Ser</t> <t>345</t> phosphorylation in S phase of the cell cycle . A . HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content ( a ) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr 345 or Chk1-Ser 345 phosphorylation status ( b ) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B . HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in ( A ). Note that very weak Vpr was detected in ( A-b ) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction ( B-b ).
    Rabbit Monoclonal Anti Phospho Chk1 Ser 345 133d3 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R"

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    Journal: Retrovirology

    doi: 10.1186/1742-4690-7-59

    Vpr induces cell cycle G2/M arrest through activation of Chk1 via Ser 345 phosphorylation in S phase of the cell cycle . A . HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content ( a ) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr 345 or Chk1-Ser 345 phosphorylation status ( b ) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B . HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in ( A ). Note that very weak Vpr was detected in ( A-b ) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction ( B-b ).
    Figure Legend Snippet: Vpr induces cell cycle G2/M arrest through activation of Chk1 via Ser 345 phosphorylation in S phase of the cell cycle . A . HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content ( a ) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr 345 or Chk1-Ser 345 phosphorylation status ( b ) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B . HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in ( A ). Note that very weak Vpr was detected in ( A-b ) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction ( B-b ).

    Techniques Used: Activation Assay, Blocking Assay, Transduction

    Chk1-Ser 345 is exclusively required for Vpr-induced G2 arrest . HeLa cells were first transfected with wild type (WT) siRNA-resistant pEGFP-Chk1 (siR-Chk1) or pEGFP-Chk1 Ser345A mutant (siR-Chk1-S345A) plasmids. The endogenous Chk1 mRNA was then depleted by a specific Chk1 siRNA for 24 hrs followed by Adv or Adv-Vpr transduction. The symbol
    Figure Legend Snippet: Chk1-Ser 345 is exclusively required for Vpr-induced G2 arrest . HeLa cells were first transfected with wild type (WT) siRNA-resistant pEGFP-Chk1 (siR-Chk1) or pEGFP-Chk1 Ser345A mutant (siR-Chk1-S345A) plasmids. The endogenous Chk1 mRNA was then depleted by a specific Chk1 siRNA for 24 hrs followed by Adv or Adv-Vpr transduction. The symbol "+" indicates presence of the siR-Chk1 or siR-Chk1-S345A plasmids. The dash sign "-"means no plasmid was introduced in wild-type Chk1, depleted by siRNA. The cell cycle profiles of the indicated cell lines were measured 48 hours after the adenoviral transduction by flow cytometric analysis ( A ). Expression of endogenous or siRNA-resistant Chk1 constructs from indicated cell lines was confirmed by Western blot analysis using anti-Chk1 antibody at the same time of flow cytometric analysis ( B ). Note that the siR-Chk1 or siR-Chk1-Ser345A gene products cannot be depleted by the normal "Chk1 siRNA" used here because silent mutations were incorporated into the Chk1 genes during site-directed mutagenesis. These silent mutations will not alter the intended protein sequences, i.e., wild type Chk1 or Chk1-Ser345A.

    Techniques Used: Transfection, Mutagenesis, Transduction, Plasmid Preparation, Expressing, Construct, Western Blot

    Chk1-Ser 345 is activated by Vpr and HU/UV with different cell cycle outcomes . Synchronized G1/S HeLa cells by DT were treated with HU, UV or transduced with Adv-Vpr at time 0, collected at the indicated time, and then subjected to Western blot analysis ( A ) using anti-Chk1-Ser 345 and anti-γH2AX-Ser 139 antibodies. The cell cycle profiles of differently treated cells were analyzed at the indicated time after the DT release by flow cytometric analysis ( B ).
    Figure Legend Snippet: Chk1-Ser 345 is activated by Vpr and HU/UV with different cell cycle outcomes . Synchronized G1/S HeLa cells by DT were treated with HU, UV or transduced with Adv-Vpr at time 0, collected at the indicated time, and then subjected to Western blot analysis ( A ) using anti-Chk1-Ser 345 and anti-γH2AX-Ser 139 antibodies. The cell cycle profiles of differently treated cells were analyzed at the indicated time after the DT release by flow cytometric analysis ( B ).

    Techniques Used: Transduction, Western Blot

    Vpr has little or no effect on proteasome-mediated protein degradation of Cdc25A in contrast to HU/UV . (A) Synchronized G1/S HeLa cells treated with HU, UV or transduced with Adv-Vpr were collected at the indicated time, and then subjected to Western blot analysis using anti-Cdc25A and anti-Vpr antibodies ( a ). β-actin was used as a loading control. The relative intensity of the Cdc25A protein levels to β-actin was determined by densitometry and the Cdc25A protein level at 0 hour was set as 1.0. ( b) . The results presented are the average of three independent experiments. ( B) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 5 hours after treatment. The protein level of Cdc25A was detected by Western blot analysis. ( C) HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by the DT blocks. HU- or Vpr-treated cells were collected 5 hours after the DT release. The protein level of Cdc25A was detected by Western blot analysis using the indicated antibodies.
    Figure Legend Snippet: Vpr has little or no effect on proteasome-mediated protein degradation of Cdc25A in contrast to HU/UV . (A) Synchronized G1/S HeLa cells treated with HU, UV or transduced with Adv-Vpr were collected at the indicated time, and then subjected to Western blot analysis using anti-Cdc25A and anti-Vpr antibodies ( a ). β-actin was used as a loading control. The relative intensity of the Cdc25A protein levels to β-actin was determined by densitometry and the Cdc25A protein level at 0 hour was set as 1.0. ( b) . The results presented are the average of three independent experiments. ( B) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 5 hours after treatment. The protein level of Cdc25A was detected by Western blot analysis. ( C) HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by the DT blocks. HU- or Vpr-treated cells were collected 5 hours after the DT release. The protein level of Cdc25A was detected by Western blot analysis using the indicated antibodies.

    Techniques Used: Transduction, Western Blot

    Vpr promotes proteasome-mediated protein degradation of Cdc25B and Cdc25C . ( A) Synchronized G1/S HeLa cells treated with HU or transduced with Adv-Vpr were collected at indicated time, and then subjected to Western blot analysis using anti-Cdc25B or anti-Cdc25C antibody, respectively ( a) . β-actin was used as a loading control. The relative intensity of the Cdc25B ( b ) or Cdc25C ( c ) protein levels to β-actin were determined by densitometry. The results presented are the average of three independent experiments. ( B) Synchronized HeLa cells were pre-treated with specific siRNA against Chk1 or treated with 50 μm MG132 at 0 hour and collected at the indicated time. The protein level of Cdc25B was detected by Western blot analysis. ( C) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 11 hours after treatment. The protein level of Cdc25C was detected by Western blot analysis ( a) . HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by DT treatment. HU or Vpr treated cells were collected 11 hours after DT release. The protein level of Cdc25C was detected by Western blot analysis using indicated antibodies ( b ).
    Figure Legend Snippet: Vpr promotes proteasome-mediated protein degradation of Cdc25B and Cdc25C . ( A) Synchronized G1/S HeLa cells treated with HU or transduced with Adv-Vpr were collected at indicated time, and then subjected to Western blot analysis using anti-Cdc25B or anti-Cdc25C antibody, respectively ( a) . β-actin was used as a loading control. The relative intensity of the Cdc25B ( b ) or Cdc25C ( c ) protein levels to β-actin were determined by densitometry. The results presented are the average of three independent experiments. ( B) Synchronized HeLa cells were pre-treated with specific siRNA against Chk1 or treated with 50 μm MG132 at 0 hour and collected at the indicated time. The protein level of Cdc25B was detected by Western blot analysis. ( C) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 11 hours after treatment. The protein level of Cdc25C was detected by Western blot analysis ( a) . HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by DT treatment. HU or Vpr treated cells were collected 11 hours after DT release. The protein level of Cdc25C was detected by Western blot analysis using indicated antibodies ( b ).

    Techniques Used: Transduction, Western Blot

    Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser 345 phosphorylation and G2 arrest in HeLa cells . (A) a . Vpr induces cellular gross enlargement (top) with single enlarged nuclei (bottom). HeLa cells were synchronized in G1/S as described. Cells were then stained with DAPI. Images were captured 11 hours after Vpr transduction using a Leica DMR fluorescence microscope (DM4500B; Leica Microsystems) equipped with a high-performance camera (Hamamatsu) under visual light (top) and fluorescence (bottom). Scale bar: 10 μm. b . Vpr promotes the accumulation of DNA polyploidy as indicated by presence of 8N DNA. HeLa cells were synchronized in G1/S as described. DNA ploidy was measured by propidium iodide staining using flow cytometric analysis over time. ( B ) Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv-Vpr at time 0 and then collected at 5 hours after viral transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser 345 antibody ( a ). The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as controls ( b ). ( C ). Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv or Adv-Vpr at time 0 and then collected at 11 hours after viral transduction for flow cytometric analysis. Ctr, control.
    Figure Legend Snippet: Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser 345 phosphorylation and G2 arrest in HeLa cells . (A) a . Vpr induces cellular gross enlargement (top) with single enlarged nuclei (bottom). HeLa cells were synchronized in G1/S as described. Cells were then stained with DAPI. Images were captured 11 hours after Vpr transduction using a Leica DMR fluorescence microscope (DM4500B; Leica Microsystems) equipped with a high-performance camera (Hamamatsu) under visual light (top) and fluorescence (bottom). Scale bar: 10 μm. b . Vpr promotes the accumulation of DNA polyploidy as indicated by presence of 8N DNA. HeLa cells were synchronized in G1/S as described. DNA ploidy was measured by propidium iodide staining using flow cytometric analysis over time. ( B ) Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv-Vpr at time 0 and then collected at 5 hours after viral transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser 345 antibody ( a ). The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as controls ( b ). ( C ). Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv or Adv-Vpr at time 0 and then collected at 11 hours after viral transduction for flow cytometric analysis. Ctr, control.

    Techniques Used: Staining, Transduction, Fluorescence, Microscopy, Western Blot

    Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser 345 phosphorylation and G2 arrest in CEM-SS cells . (A) Vpr promotes accumulation of DNA polyploidy as indicated by the presence of 8N DNA. Asynchronized CEM-SS cells were grown under the normal cell culture condition, and transduced with Adv viral control or Adv-Vpr. Cells were collected at indicated time point and DNA ploidy was measured by PI staining using flow cytometric analysis. ( B ) Asynchronized CEM-SS cells were pretreated with Cdt1, Cdc6 or control (Ctr) siRNA, and then transduced with Adv or Adv-Vpr 24 hours after addition of siRNAs. Cells were then harvested 48 hours post-transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser 345 antibody. The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as protein loading controls. ( C ). CEM-SS were treated the same way as described in ( B ). The cells were harvested 48 hours post-transduction and the cell lysates were then subjected to flow cytometric analysis.
    Figure Legend Snippet: Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser 345 phosphorylation and G2 arrest in CEM-SS cells . (A) Vpr promotes accumulation of DNA polyploidy as indicated by the presence of 8N DNA. Asynchronized CEM-SS cells were grown under the normal cell culture condition, and transduced with Adv viral control or Adv-Vpr. Cells were collected at indicated time point and DNA ploidy was measured by PI staining using flow cytometric analysis. ( B ) Asynchronized CEM-SS cells were pretreated with Cdt1, Cdc6 or control (Ctr) siRNA, and then transduced with Adv or Adv-Vpr 24 hours after addition of siRNAs. Cells were then harvested 48 hours post-transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser 345 antibody. The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as protein loading controls. ( C ). CEM-SS were treated the same way as described in ( B ). The cells were harvested 48 hours post-transduction and the cell lysates were then subjected to flow cytometric analysis.

    Techniques Used: Cell Culture, Transduction, Staining, Western Blot

    anti phospho chk1 ser 345  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho chk1 ser 345
    Anti 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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    phospho ser 345 chk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho ser 345 chk1
    PARP inhibition activates ATM. ( A ) Western blot for ATM phospho serine 1981 and ATM control following PARP inhibition for the times indicated. ( B ) Western blot for <t>CHK1</t> phospho serine 345, CHK2 phospho threonine 68 and total CHK2 and actin controls following PARP inhibition or 0.5 mM HU treatment for 24 h.
    Phospho Ser 345 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 "Inhibition of poly (ADP-ribose) polymerase activates ATM which is required for subsequent homologous recombination repair"

    Article Title: Inhibition of poly (ADP-ribose) polymerase activates ATM which is required for subsequent homologous recombination repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl108

    PARP inhibition activates ATM. ( A ) Western blot for ATM phospho serine 1981 and ATM control following PARP inhibition for the times indicated. ( B ) Western blot for CHK1 phospho serine 345, CHK2 phospho threonine 68 and total CHK2 and actin controls following PARP inhibition or 0.5 mM HU treatment for 24 h.
    Figure Legend Snippet: PARP inhibition activates ATM. ( A ) Western blot for ATM phospho serine 1981 and ATM control following PARP inhibition for the times indicated. ( B ) Western blot for CHK1 phospho serine 345, CHK2 phospho threonine 68 and total CHK2 and actin controls following PARP inhibition or 0.5 mM HU treatment for 24 h.

    Techniques Used: Inhibition, Western Blot

    phospho chk1 ser 345  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho chk1 ser 345
    Cell cycle analysis was performed by flow cytometry. Representative cell cycle profiles of A549 cells treated with 50, 100, 200-A represents the intensity of propidium iodide, and the y axis represents the cell counts (A). Concentration-dependent effect of Cuc B was evaluated by flow cytometer to quantitate the number of cells in G2/M phase in A549 cells. Values are means ± S.E.M of three independent experiments, each conducted in triplicate (B). Western blot analyses of ATM, <t>Chk1,</t> Chk2, Cdc25C, p53, 14-3-3-σ, Cdk1, Cyclin B1 and their phophorylated forms in Cuc B exposed A549 cells for 24 h (C-E). Total 14-3-3-σ protein was immunoprecipitated from control and Cuc B treated A549 cells lysates and analyzed for p-Cdc25C (Ser-216) for 24 h (F). ** p <0.01 vs. Cont. Cont, control group.
    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 "Cucurbitacin B Induced ATM-Mediated DNA Damage Causes G2/M Cell Cycle Arrest in a ROS-Dependent Manner"

    Article Title: Cucurbitacin B Induced ATM-Mediated DNA Damage Causes G2/M Cell Cycle Arrest in a ROS-Dependent Manner

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088140

    Cell cycle analysis was performed by flow cytometry. Representative cell cycle profiles of A549 cells treated with 50, 100, 200-A represents the intensity of propidium iodide, and the y axis represents the cell counts (A). Concentration-dependent effect of Cuc B was evaluated by flow cytometer to quantitate the number of cells in G2/M phase in A549 cells. Values are means ± S.E.M of three independent experiments, each conducted in triplicate (B). Western blot analyses of ATM, Chk1, Chk2, Cdc25C, p53, 14-3-3-σ, Cdk1, Cyclin B1 and their phophorylated forms in Cuc B exposed A549 cells for 24 h (C-E). Total 14-3-3-σ protein was immunoprecipitated from control and Cuc B treated A549 cells lysates and analyzed for p-Cdc25C (Ser-216) for 24 h (F). ** p <0.01 vs. Cont. Cont, control group.
    Figure Legend Snippet: Cell cycle analysis was performed by flow cytometry. Representative cell cycle profiles of A549 cells treated with 50, 100, 200-A represents the intensity of propidium iodide, and the y axis represents the cell counts (A). Concentration-dependent effect of Cuc B was evaluated by flow cytometer to quantitate the number of cells in G2/M phase in A549 cells. Values are means ± S.E.M of three independent experiments, each conducted in triplicate (B). Western blot analyses of ATM, Chk1, Chk2, Cdc25C, p53, 14-3-3-σ, Cdk1, Cyclin B1 and their phophorylated forms in Cuc B exposed A549 cells for 24 h (C-E). Total 14-3-3-σ protein was immunoprecipitated from control and Cuc B treated A549 cells lysates and analyzed for p-Cdc25C (Ser-216) for 24 h (F). ** p <0.01 vs. Cont. Cont, control group.

    Techniques Used: Cell Cycle Assay, Flow Cytometry, Concentration Assay, Western Blot, Immunoprecipitation

    A549 cells transfected with 100(A, B). A549 cells transfected with ATM siRNA and treated with or without 200 nM Cuc B for 24 h. Phosphorylation of Chk1 on Ser-345, Cdc25C on Ser-216, p53 on Ser-15 and protein levels of p53 Cyclin B1 were analyzed by Western blot (C). Total Cyclin B1 protein was immunoprecipitated from ATM siRNA transfected and treated with or without 200 nM Cuc B A549 cells lysates and analyzed for Cdk1 for 24 h (D). * p <0.05 vs. Cont. Cont, control group.
    Figure Legend Snippet: A549 cells transfected with 100(A, B). A549 cells transfected with ATM siRNA and treated with or without 200 nM Cuc B for 24 h. Phosphorylation of Chk1 on Ser-345, Cdc25C on Ser-216, p53 on Ser-15 and protein levels of p53 Cyclin B1 were analyzed by Western blot (C). Total Cyclin B1 protein was immunoprecipitated from ATM siRNA transfected and treated with or without 200 nM Cuc B A549 cells lysates and analyzed for Cdk1 for 24 h (D). * p <0.05 vs. Cont. Cont, control group.

    Techniques Used: Transfection, Western Blot, Immunoprecipitation

    A549 cells transfected with 100(A, B). A549 cells transfected with Chk1 siRNA and treated with or without 200 nM Cuc B for 24 h. Phosphorylation of Cdc25C on Ser-216, Cdk1 on were analyzed by Western blot (C). * p <0.05 vs. Cont. Cont, control group.
    Figure Legend Snippet: A549 cells transfected with 100(A, B). A549 cells transfected with Chk1 siRNA and treated with or without 200 nM Cuc B for 24 h. Phosphorylation of Cdc25C on Ser-216, Cdk1 on were analyzed by Western blot (C). * p <0.05 vs. Cont. Cont, control group.

    Techniques Used: Transfection, Western Blot

    The generation of ROS in A549 cells after 50, 100, 200 2 -DA as described under Materials and Methods (A, B). Effect of Cuc B on STAT3 phosphorylation on Tyr-705 and STAT3 expression were analyzed by Western blot assay (C). A549 cells were treated with 10 mM NAC for 0.5 h followed by treatment with 200 nM Cuc B for 24 h, and the cell cycle was tested (D, E). A549 cells pretreated with 10 mM NAC for 0.5 h and treated with or without 200 nM Cuc B for 24 h. Phosphorylation of Chk1 on Ser-345, Cdc25C on Ser-216, p53 on Ser-15 and protein levels of Chk1, Cdc25C, p53, 14-3-3-σ, Cdk1 were analyzed by Western blot assay (F). * p <0.05 vs. Cont, *** p <0.001 vs. Cont. Cont, control group.
    Figure Legend Snippet: The generation of ROS in A549 cells after 50, 100, 200 2 -DA as described under Materials and Methods (A, B). Effect of Cuc B on STAT3 phosphorylation on Tyr-705 and STAT3 expression were analyzed by Western blot assay (C). A549 cells were treated with 10 mM NAC for 0.5 h followed by treatment with 200 nM Cuc B for 24 h, and the cell cycle was tested (D, E). A549 cells pretreated with 10 mM NAC for 0.5 h and treated with or without 200 nM Cuc B for 24 h. Phosphorylation of Chk1 on Ser-345, Cdc25C on Ser-216, p53 on Ser-15 and protein levels of Chk1, Cdc25C, p53, 14-3-3-σ, Cdk1 were analyzed by Western blot assay (F). * p <0.05 vs. Cont, *** p <0.001 vs. Cont. Cont, control group.

    Techniques Used: Expressing, Western Blot

    Cuc B induced ROS formation, which lead to DNA damage. In response to DNA damage, ATM via autophosphorylation activated G2/M checkpoint. The downstream effectors of ATM, Chk1 and p53, were both activated, which resulted in Cdc25C phosphorylation and increased expression of 14-3-3-σ respectively. Phosphorylation of Cdc25C on Ser-216 by activated Chk1 facilitated the binding of 14-3-3-σ to Cdc25C, which prevents Cdk1 dephosphorylation, and keeps Cdk1-Cyclin B1 complex inactivated, causing G2/M phase arrest.
    Figure Legend Snippet: Cuc B induced ROS formation, which lead to DNA damage. In response to DNA damage, ATM via autophosphorylation activated G2/M checkpoint. The downstream effectors of ATM, Chk1 and p53, were both activated, which resulted in Cdc25C phosphorylation and increased expression of 14-3-3-σ respectively. Phosphorylation of Cdc25C on Ser-216 by activated Chk1 facilitated the binding of 14-3-3-σ to Cdc25C, which prevents Cdk1 dephosphorylation, and keeps Cdk1-Cyclin B1 complex inactivated, causing G2/M phase arrest.

    Techniques Used: Expressing, Binding Assay, De-Phosphorylation Assay

    phosphorylated ser 345 chk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phosphorylated ser 345 chk1
    (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of <t>pCHK1/CHK1</t> and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.
    Phosphorylated Ser 345 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 "Mutation of Serine 1333 in the ATR HEAT Repeats Creates a Hyperactive Kinase"

    Article Title: Mutation of Serine 1333 in the ATR HEAT Repeats Creates a Hyperactive Kinase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0099397

    (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of pCHK1/CHK1 and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.
    Figure Legend Snippet: (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of pCHK1/CHK1 and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.

    Techniques Used: Clone Assay, Expressing, SDS Page, Western Blot

    rabbit ser 345 p chk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit ser 345 p chk1
    Real-time PCR analysis of RRM2 ( A , D ) gene expression and number of surviving cells ( B , E ) under RRM2 siRNA knockdown for NCI-H295R and MUC-1, respectively. RRM2 gene expression upon etoposide and doxorubicin treatment for NCI-H295R and MUC-1 ( C , F ). Quantification of Western blots for <t>p-Chk1</t> ( G ), p-Chk2 ( H ), and p-H2AX ( I ) for no treatment, gemcitabine, cisplatin, and combination of both (gemcitabine and cisplatin) 24 h after treatment. Schematic illustration of the related DNA damage–repair pathway, including relevant therapeutic inhibitors ( J ). Stars represent significance vs. nontreated (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001); ns, non-significant.
    Rabbit Ser 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 "Novel Insights into the Molecular Regulation of Ribonucleotide Reductase in Adrenocortical Carcinoma Treatment"

    Article Title: Novel Insights into the Molecular Regulation of Ribonucleotide Reductase in Adrenocortical Carcinoma Treatment

    Journal: Cancers

    doi: 10.3390/cancers13164200

    Real-time PCR analysis of RRM2 ( A , D ) gene expression and number of surviving cells ( B , E ) under RRM2 siRNA knockdown for NCI-H295R and MUC-1, respectively. RRM2 gene expression upon etoposide and doxorubicin treatment for NCI-H295R and MUC-1 ( C , F ). Quantification of Western blots for p-Chk1 ( G ), p-Chk2 ( H ), and p-H2AX ( I ) for no treatment, gemcitabine, cisplatin, and combination of both (gemcitabine and cisplatin) 24 h after treatment. Schematic illustration of the related DNA damage–repair pathway, including relevant therapeutic inhibitors ( J ). Stars represent significance vs. nontreated (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001); ns, non-significant.
    Figure Legend Snippet: Real-time PCR analysis of RRM2 ( A , D ) gene expression and number of surviving cells ( B , E ) under RRM2 siRNA knockdown for NCI-H295R and MUC-1, respectively. RRM2 gene expression upon etoposide and doxorubicin treatment for NCI-H295R and MUC-1 ( C , F ). Quantification of Western blots for p-Chk1 ( G ), p-Chk2 ( H ), and p-H2AX ( I ) for no treatment, gemcitabine, cisplatin, and combination of both (gemcitabine and cisplatin) 24 h after treatment. Schematic illustration of the related DNA damage–repair pathway, including relevant therapeutic inhibitors ( J ). Stars represent significance vs. nontreated (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001); ns, non-significant.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Western Blot

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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 phospho chk1 ser 345
    siRNA screen for genes that promote proliferation of MDA-MB-231 cells identify <t>CHK1,</t> RRM1 and RRM2 as top-hits . ( A ) Effects on proliferation by gene knockdown with the custom siRNA library. Data are shown as a z-score distribution from the mean. ( B ) Percent change (from non-targeting siRNA (NTS) control) in proliferation of cells due to knock-down of expression by individual siRNA oligos for the genes noted. Q-RT-PCR determination of reduced RNA ( C ) and protein ( D ) expression for CHK1, RRMI, and RRM2 for individual siRNA oligos. (E) Percent growth of cells due to expression of Qiagen CHK1-siRNA. Gene ( F ) and protein ( G ) expression for CHK1 is suppressed in cells that are transfected with individual Qiagen CHK1-siRNAs. Graphs shown are representative of 3 repeated experiments.
    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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    Vpr induces cell cycle G2/M arrest through activation of <t>Chk1</t> via <t>Ser</t> <t>345</t> phosphorylation in S phase of the cell cycle . A . HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content ( a ) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr 345 or Chk1-Ser 345 phosphorylation status ( b ) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B . HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in ( A ). Note that very weak Vpr was detected in ( A-b ) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction ( B-b ).
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    Vpr induces cell cycle G2/M arrest through activation of <t>Chk1</t> via <t>Ser</t> <t>345</t> phosphorylation in S phase of the cell cycle . A . HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content ( a ) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr 345 or Chk1-Ser 345 phosphorylation status ( b ) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B . HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in ( A ). Note that very weak Vpr was detected in ( A-b ) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction ( B-b ).
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    Cell Signaling Technology Inc phospho ser 345 chk1
    PARP inhibition activates ATM. ( A ) Western blot for ATM phospho serine 1981 and ATM control following PARP inhibition for the times indicated. ( B ) Western blot for <t>CHK1</t> phospho serine 345, CHK2 phospho threonine 68 and total CHK2 and actin controls following PARP inhibition or 0.5 mM HU treatment for 24 h.
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    (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of <t>pCHK1/CHK1</t> and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.
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    Cell Signaling Technology Inc rabbit ser 345 p chk1
    Real-time PCR analysis of RRM2 ( A , D ) gene expression and number of surviving cells ( B , E ) under RRM2 siRNA knockdown for NCI-H295R and MUC-1, respectively. RRM2 gene expression upon etoposide and doxorubicin treatment for NCI-H295R and MUC-1 ( C , F ). Quantification of Western blots for <t>p-Chk1</t> ( G ), p-Chk2 ( H ), and p-H2AX ( I ) for no treatment, gemcitabine, cisplatin, and combination of both (gemcitabine and cisplatin) 24 h after treatment. Schematic illustration of the related DNA damage–repair pathway, including relevant therapeutic inhibitors ( J ). Stars represent significance vs. nontreated (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001); ns, non-significant.
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    Image Search Results


    ( 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

    siRNA screen for genes that promote proliferation of MDA-MB-231 cells identify CHK1, RRM1 and RRM2 as top-hits . ( A ) Effects on proliferation by gene knockdown with the custom siRNA library. Data are shown as a z-score distribution from the mean. ( B ) Percent change (from non-targeting siRNA (NTS) control) in proliferation of cells due to knock-down of expression by individual siRNA oligos for the genes noted. Q-RT-PCR determination of reduced RNA ( C ) and protein ( D ) expression for CHK1, RRMI, and RRM2 for individual siRNA oligos. (E) Percent growth of cells due to expression of Qiagen CHK1-siRNA. Gene ( F ) and protein ( G ) expression for CHK1 is suppressed in cells that are transfected with individual Qiagen CHK1-siRNAs. Graphs shown are representative of 3 repeated experiments.

    Journal: Breast Cancer Research : BCR

    Article Title: Cross-species genomic and functional analyses identify a combination therapy using a CHK1 inhibitor and a ribonucleotide reductase inhibitor to treat triple-negative breast cancer

    doi: 10.1186/bcr3230

    Figure Lengend Snippet: siRNA screen for genes that promote proliferation of MDA-MB-231 cells identify CHK1, RRM1 and RRM2 as top-hits . ( A ) Effects on proliferation by gene knockdown with the custom siRNA library. Data are shown as a z-score distribution from the mean. ( B ) Percent change (from non-targeting siRNA (NTS) control) in proliferation of cells due to knock-down of expression by individual siRNA oligos for the genes noted. Q-RT-PCR determination of reduced RNA ( C ) and protein ( D ) expression for CHK1, RRMI, and RRM2 for individual siRNA oligos. (E) Percent growth of cells due to expression of Qiagen CHK1-siRNA. Gene ( F ) and protein ( G ) expression for CHK1 is suppressed in cells that are transfected with individual Qiagen CHK1-siRNAs. Graphs shown are representative of 3 repeated experiments.

    Article Snippet: Protein was quantified using a BCA protein assay kit (Pierce, Rockford, IL), separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Invitrogen) for detection using the following primary antibodies: RRM1 (3388; Cell Signal, Danvers, MA, USA), RRM2 (10846; Santa Cruz, Santa Cruz, CA, USA), β-tubublin (RB-9249; Thermo Scientific, Rockford, IL, USA ), Phospho-gamma H2AX (2577; Cell Signaling Technology, Beverly, MA), Cyclin A (Rb-1548; Neomarkers, Freemont, CA, USA) β-actin (A1978; Sigma), phospho Chk1 ser-345 (Rb-2348; Cell Signaling Technology), Chk1 (Ms-2360; Cell Signaling Technology).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection

    Combination therapy with CHK1 inhibitors and gemcitabine inhibits proliferation in TNBC cells . MDA-MB-231 ( A, C ) and M6 ( B, D ) cells were treated with agents on day 0 and proliferation was measured on days indicated. MDA-MB-231 cells (gemcitabine 10 nM; UCN-01 150 nM), (gemcitabine10 nM; AZD 7762 200 nM). M6 cells (gemcitabine 4 nM; UCN-01 20 nM) (gemcitabine 4 nM; AZD 7762 30 nM). Results in B and D are from one experiment thus Gem treatment and vehicle are the same in both panels. P -value based upon change from vehicle treatment (letters only) and from single agent to combination treatment (line and letter) (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).

    Journal: Breast Cancer Research : BCR

    Article Title: Cross-species genomic and functional analyses identify a combination therapy using a CHK1 inhibitor and a ribonucleotide reductase inhibitor to treat triple-negative breast cancer

    doi: 10.1186/bcr3230

    Figure Lengend Snippet: Combination therapy with CHK1 inhibitors and gemcitabine inhibits proliferation in TNBC cells . MDA-MB-231 ( A, C ) and M6 ( B, D ) cells were treated with agents on day 0 and proliferation was measured on days indicated. MDA-MB-231 cells (gemcitabine 10 nM; UCN-01 150 nM), (gemcitabine10 nM; AZD 7762 200 nM). M6 cells (gemcitabine 4 nM; UCN-01 20 nM) (gemcitabine 4 nM; AZD 7762 30 nM). Results in B and D are from one experiment thus Gem treatment and vehicle are the same in both panels. P -value based upon change from vehicle treatment (letters only) and from single agent to combination treatment (line and letter) (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).

    Article Snippet: Protein was quantified using a BCA protein assay kit (Pierce, Rockford, IL), separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Invitrogen) for detection using the following primary antibodies: RRM1 (3388; Cell Signal, Danvers, MA, USA), RRM2 (10846; Santa Cruz, Santa Cruz, CA, USA), β-tubublin (RB-9249; Thermo Scientific, Rockford, IL, USA ), Phospho-gamma H2AX (2577; Cell Signaling Technology, Beverly, MA), Cyclin A (Rb-1548; Neomarkers, Freemont, CA, USA) β-actin (A1978; Sigma), phospho Chk1 ser-345 (Rb-2348; Cell Signaling Technology), Chk1 (Ms-2360; Cell Signaling Technology).

    Techniques:

    Combination therapy with UCN-01 and gemcitabine induces DNA damage and apoptosis in TNBC cells . ( A ) Protein samples were collected 24 hours after drug treatment to detect changes in DNA damage (gamma-H2AX), checkpoint activation (phos-CHK1 andtTotal CHK1) and cell cycle progression (Cyclin A) by immunoblot analysis. ( B ) Cell cycle changes were assessed by BrdU-labeling and propidium iodide staining 24 hour after drug treatment. ( C ) Percentage of cells at 48 hours in early apoptosis (Annexin V + /7-AAD - ) and late apoptosis (Annexin V+/7-AAD+) with representative data ( D ). MDA-MB-231 cells (gemcitabine 10 nM; UCN-01 150 nM), M6 cells (gemcitabine 4 nM; UCN-01 20 nM). P -value based upon change from vehicle treatment (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).

    Journal: Breast Cancer Research : BCR

    Article Title: Cross-species genomic and functional analyses identify a combination therapy using a CHK1 inhibitor and a ribonucleotide reductase inhibitor to treat triple-negative breast cancer

    doi: 10.1186/bcr3230

    Figure Lengend Snippet: Combination therapy with UCN-01 and gemcitabine induces DNA damage and apoptosis in TNBC cells . ( A ) Protein samples were collected 24 hours after drug treatment to detect changes in DNA damage (gamma-H2AX), checkpoint activation (phos-CHK1 andtTotal CHK1) and cell cycle progression (Cyclin A) by immunoblot analysis. ( B ) Cell cycle changes were assessed by BrdU-labeling and propidium iodide staining 24 hour after drug treatment. ( C ) Percentage of cells at 48 hours in early apoptosis (Annexin V + /7-AAD - ) and late apoptosis (Annexin V+/7-AAD+) with representative data ( D ). MDA-MB-231 cells (gemcitabine 10 nM; UCN-01 150 nM), M6 cells (gemcitabine 4 nM; UCN-01 20 nM). P -value based upon change from vehicle treatment (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).

    Article Snippet: Protein was quantified using a BCA protein assay kit (Pierce, Rockford, IL), separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Invitrogen) for detection using the following primary antibodies: RRM1 (3388; Cell Signal, Danvers, MA, USA), RRM2 (10846; Santa Cruz, Santa Cruz, CA, USA), β-tubublin (RB-9249; Thermo Scientific, Rockford, IL, USA ), Phospho-gamma H2AX (2577; Cell Signaling Technology, Beverly, MA), Cyclin A (Rb-1548; Neomarkers, Freemont, CA, USA) β-actin (A1978; Sigma), phospho Chk1 ser-345 (Rb-2348; Cell Signaling Technology), Chk1 (Ms-2360; Cell Signaling Technology).

    Techniques: Activation Assay, Western Blot, Labeling, Staining

    Combination therapy with CHK1 inhibitors and gemcitabine inhibits proliferation in TNBC cells . BT-549 ( A ), SUM 159 ( B) and HCC 1187 (C ) cells were treated with agents on day 0 and proliferation was measured on days indicated. BT-549 cells (gemcitabine 10 nM; UCN-01 100 nM; AZD 7762 150 nM). SUM 159 (gemcitabine 4 nM; UCN-01 80 nM; AZD 7762 300 nM). HCC 1187 (gemcitabine 10 nM; UCN-01 150 nM; AZD 7762 200 nM). P -value based upon change from vehicle treatment (letters only) and from single agent to combination treatment (line and letter; day three only) (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).

    Journal: Breast Cancer Research : BCR

    Article Title: Cross-species genomic and functional analyses identify a combination therapy using a CHK1 inhibitor and a ribonucleotide reductase inhibitor to treat triple-negative breast cancer

    doi: 10.1186/bcr3230

    Figure Lengend Snippet: Combination therapy with CHK1 inhibitors and gemcitabine inhibits proliferation in TNBC cells . BT-549 ( A ), SUM 159 ( B) and HCC 1187 (C ) cells were treated with agents on day 0 and proliferation was measured on days indicated. BT-549 cells (gemcitabine 10 nM; UCN-01 100 nM; AZD 7762 150 nM). SUM 159 (gemcitabine 4 nM; UCN-01 80 nM; AZD 7762 300 nM). HCC 1187 (gemcitabine 10 nM; UCN-01 150 nM; AZD 7762 200 nM). P -value based upon change from vehicle treatment (letters only) and from single agent to combination treatment (line and letter; day three only) (A ≤ 0.01, B ≤ 0.005, C ≤ 0.001, D ≤ 0.0005).

    Article Snippet: Protein was quantified using a BCA protein assay kit (Pierce, Rockford, IL), separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Invitrogen) for detection using the following primary antibodies: RRM1 (3388; Cell Signal, Danvers, MA, USA), RRM2 (10846; Santa Cruz, Santa Cruz, CA, USA), β-tubublin (RB-9249; Thermo Scientific, Rockford, IL, USA ), Phospho-gamma H2AX (2577; Cell Signaling Technology, Beverly, MA), Cyclin A (Rb-1548; Neomarkers, Freemont, CA, USA) β-actin (A1978; Sigma), phospho Chk1 ser-345 (Rb-2348; Cell Signaling Technology), Chk1 (Ms-2360; Cell Signaling Technology).

    Techniques:

    In vivo response of TNBC tumors to agents that target CHK1, RRM1 and RRM2 . ( A ) SCID mice with MDA-MB-231 tumor xenografts were treated seven days after implantation with vehicle, 5 mg/kg gemcitabine, 6 mg/kg UCN-01, or a combination of both on a Q4Dx3 schedule. Gemcitabine was delivered first by IP injection and UCN-01 was delivered 24 hours later by IV injection on a Q6Hx2. Note: Inset graph highlights days 7 to 21. During this period, mice treated with the combination therapy had slower growing tumors than those treated only with gemcitabine. ( B ) SCID mice with C3(1)Tag tumor transplants were dosed 12 days after transplantation with 20 mg/kg gemcitabine, 4.5 mg/kg UCN-01, or the drug combination on a Q4Dx3 schedule. Gemcitabine was delivered first and UCN-01 was delivered eight hours later on a Q6Hx2 schedule.

    Journal: Breast Cancer Research : BCR

    Article Title: Cross-species genomic and functional analyses identify a combination therapy using a CHK1 inhibitor and a ribonucleotide reductase inhibitor to treat triple-negative breast cancer

    doi: 10.1186/bcr3230

    Figure Lengend Snippet: In vivo response of TNBC tumors to agents that target CHK1, RRM1 and RRM2 . ( A ) SCID mice with MDA-MB-231 tumor xenografts were treated seven days after implantation with vehicle, 5 mg/kg gemcitabine, 6 mg/kg UCN-01, or a combination of both on a Q4Dx3 schedule. Gemcitabine was delivered first by IP injection and UCN-01 was delivered 24 hours later by IV injection on a Q6Hx2. Note: Inset graph highlights days 7 to 21. During this period, mice treated with the combination therapy had slower growing tumors than those treated only with gemcitabine. ( B ) SCID mice with C3(1)Tag tumor transplants were dosed 12 days after transplantation with 20 mg/kg gemcitabine, 4.5 mg/kg UCN-01, or the drug combination on a Q4Dx3 schedule. Gemcitabine was delivered first and UCN-01 was delivered eight hours later on a Q6Hx2 schedule.

    Article Snippet: Protein was quantified using a BCA protein assay kit (Pierce, Rockford, IL), separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Invitrogen) for detection using the following primary antibodies: RRM1 (3388; Cell Signal, Danvers, MA, USA), RRM2 (10846; Santa Cruz, Santa Cruz, CA, USA), β-tubublin (RB-9249; Thermo Scientific, Rockford, IL, USA ), Phospho-gamma H2AX (2577; Cell Signaling Technology, Beverly, MA), Cyclin A (Rb-1548; Neomarkers, Freemont, CA, USA) β-actin (A1978; Sigma), phospho Chk1 ser-345 (Rb-2348; Cell Signaling Technology), Chk1 (Ms-2360; Cell Signaling Technology).

    Techniques: In Vivo, Injection, IV Injection, Transplantation Assay

    Vpr induces cell cycle G2/M arrest through activation of Chk1 via Ser 345 phosphorylation in S phase of the cell cycle . A . HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content ( a ) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr 345 or Chk1-Ser 345 phosphorylation status ( b ) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B . HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in ( A ). Note that very weak Vpr was detected in ( A-b ) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction ( B-b ).

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Vpr induces cell cycle G2/M arrest through activation of Chk1 via Ser 345 phosphorylation in S phase of the cell cycle . A . HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content ( a ) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr 345 or Chk1-Ser 345 phosphorylation status ( b ) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B . HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in ( A ). Note that very weak Vpr was detected in ( A-b ) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction ( B-b ).

    Article Snippet: Rabbit monoclonal anti-phospho-Chk1-Ser 345 (133D3) antibody was purchased from Cell Signaling Technology, Inc (Danvers, MA).

    Techniques: Activation Assay, Blocking Assay, Transduction

    Chk1-Ser 345 is exclusively required for Vpr-induced G2 arrest . HeLa cells were first transfected with wild type (WT) siRNA-resistant pEGFP-Chk1 (siR-Chk1) or pEGFP-Chk1 Ser345A mutant (siR-Chk1-S345A) plasmids. The endogenous Chk1 mRNA was then depleted by a specific Chk1 siRNA for 24 hrs followed by Adv or Adv-Vpr transduction. The symbol

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Chk1-Ser 345 is exclusively required for Vpr-induced G2 arrest . HeLa cells were first transfected with wild type (WT) siRNA-resistant pEGFP-Chk1 (siR-Chk1) or pEGFP-Chk1 Ser345A mutant (siR-Chk1-S345A) plasmids. The endogenous Chk1 mRNA was then depleted by a specific Chk1 siRNA for 24 hrs followed by Adv or Adv-Vpr transduction. The symbol "+" indicates presence of the siR-Chk1 or siR-Chk1-S345A plasmids. The dash sign "-"means no plasmid was introduced in wild-type Chk1, depleted by siRNA. The cell cycle profiles of the indicated cell lines were measured 48 hours after the adenoviral transduction by flow cytometric analysis ( A ). Expression of endogenous or siRNA-resistant Chk1 constructs from indicated cell lines was confirmed by Western blot analysis using anti-Chk1 antibody at the same time of flow cytometric analysis ( B ). Note that the siR-Chk1 or siR-Chk1-Ser345A gene products cannot be depleted by the normal "Chk1 siRNA" used here because silent mutations were incorporated into the Chk1 genes during site-directed mutagenesis. These silent mutations will not alter the intended protein sequences, i.e., wild type Chk1 or Chk1-Ser345A.

    Article Snippet: Rabbit monoclonal anti-phospho-Chk1-Ser 345 (133D3) antibody was purchased from Cell Signaling Technology, Inc (Danvers, MA).

    Techniques: Transfection, Mutagenesis, Transduction, Plasmid Preparation, Expressing, Construct, Western Blot

    Chk1-Ser 345 is activated by Vpr and HU/UV with different cell cycle outcomes . Synchronized G1/S HeLa cells by DT were treated with HU, UV or transduced with Adv-Vpr at time 0, collected at the indicated time, and then subjected to Western blot analysis ( A ) using anti-Chk1-Ser 345 and anti-γH2AX-Ser 139 antibodies. The cell cycle profiles of differently treated cells were analyzed at the indicated time after the DT release by flow cytometric analysis ( B ).

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Chk1-Ser 345 is activated by Vpr and HU/UV with different cell cycle outcomes . Synchronized G1/S HeLa cells by DT were treated with HU, UV or transduced with Adv-Vpr at time 0, collected at the indicated time, and then subjected to Western blot analysis ( A ) using anti-Chk1-Ser 345 and anti-γH2AX-Ser 139 antibodies. The cell cycle profiles of differently treated cells were analyzed at the indicated time after the DT release by flow cytometric analysis ( B ).

    Article Snippet: Rabbit monoclonal anti-phospho-Chk1-Ser 345 (133D3) antibody was purchased from Cell Signaling Technology, Inc (Danvers, MA).

    Techniques: Transduction, Western Blot

    Vpr has little or no effect on proteasome-mediated protein degradation of Cdc25A in contrast to HU/UV . (A) Synchronized G1/S HeLa cells treated with HU, UV or transduced with Adv-Vpr were collected at the indicated time, and then subjected to Western blot analysis using anti-Cdc25A and anti-Vpr antibodies ( a ). β-actin was used as a loading control. The relative intensity of the Cdc25A protein levels to β-actin was determined by densitometry and the Cdc25A protein level at 0 hour was set as 1.0. ( b) . The results presented are the average of three independent experiments. ( B) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 5 hours after treatment. The protein level of Cdc25A was detected by Western blot analysis. ( C) HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by the DT blocks. HU- or Vpr-treated cells were collected 5 hours after the DT release. The protein level of Cdc25A was detected by Western blot analysis using the indicated antibodies.

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Vpr has little or no effect on proteasome-mediated protein degradation of Cdc25A in contrast to HU/UV . (A) Synchronized G1/S HeLa cells treated with HU, UV or transduced with Adv-Vpr were collected at the indicated time, and then subjected to Western blot analysis using anti-Cdc25A and anti-Vpr antibodies ( a ). β-actin was used as a loading control. The relative intensity of the Cdc25A protein levels to β-actin was determined by densitometry and the Cdc25A protein level at 0 hour was set as 1.0. ( b) . The results presented are the average of three independent experiments. ( B) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 5 hours after treatment. The protein level of Cdc25A was detected by Western blot analysis. ( C) HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by the DT blocks. HU- or Vpr-treated cells were collected 5 hours after the DT release. The protein level of Cdc25A was detected by Western blot analysis using the indicated antibodies.

    Article Snippet: Rabbit monoclonal anti-phospho-Chk1-Ser 345 (133D3) antibody was purchased from Cell Signaling Technology, Inc (Danvers, MA).

    Techniques: Transduction, Western Blot

    Vpr promotes proteasome-mediated protein degradation of Cdc25B and Cdc25C . ( A) Synchronized G1/S HeLa cells treated with HU or transduced with Adv-Vpr were collected at indicated time, and then subjected to Western blot analysis using anti-Cdc25B or anti-Cdc25C antibody, respectively ( a) . β-actin was used as a loading control. The relative intensity of the Cdc25B ( b ) or Cdc25C ( c ) protein levels to β-actin were determined by densitometry. The results presented are the average of three independent experiments. ( B) Synchronized HeLa cells were pre-treated with specific siRNA against Chk1 or treated with 50 μm MG132 at 0 hour and collected at the indicated time. The protein level of Cdc25B was detected by Western blot analysis. ( C) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 11 hours after treatment. The protein level of Cdc25C was detected by Western blot analysis ( a) . HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by DT treatment. HU or Vpr treated cells were collected 11 hours after DT release. The protein level of Cdc25C was detected by Western blot analysis using indicated antibodies ( b ).

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Vpr promotes proteasome-mediated protein degradation of Cdc25B and Cdc25C . ( A) Synchronized G1/S HeLa cells treated with HU or transduced with Adv-Vpr were collected at indicated time, and then subjected to Western blot analysis using anti-Cdc25B or anti-Cdc25C antibody, respectively ( a) . β-actin was used as a loading control. The relative intensity of the Cdc25B ( b ) or Cdc25C ( c ) protein levels to β-actin were determined by densitometry. The results presented are the average of three independent experiments. ( B) Synchronized HeLa cells were pre-treated with specific siRNA against Chk1 or treated with 50 μm MG132 at 0 hour and collected at the indicated time. The protein level of Cdc25B was detected by Western blot analysis. ( C) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 11 hours after treatment. The protein level of Cdc25C was detected by Western blot analysis ( a) . HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by DT treatment. HU or Vpr treated cells were collected 11 hours after DT release. The protein level of Cdc25C was detected by Western blot analysis using indicated antibodies ( b ).

    Article Snippet: Rabbit monoclonal anti-phospho-Chk1-Ser 345 (133D3) antibody was purchased from Cell Signaling Technology, Inc (Danvers, MA).

    Techniques: Transduction, Western Blot

    Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser 345 phosphorylation and G2 arrest in HeLa cells . (A) a . Vpr induces cellular gross enlargement (top) with single enlarged nuclei (bottom). HeLa cells were synchronized in G1/S as described. Cells were then stained with DAPI. Images were captured 11 hours after Vpr transduction using a Leica DMR fluorescence microscope (DM4500B; Leica Microsystems) equipped with a high-performance camera (Hamamatsu) under visual light (top) and fluorescence (bottom). Scale bar: 10 μm. b . Vpr promotes the accumulation of DNA polyploidy as indicated by presence of 8N DNA. HeLa cells were synchronized in G1/S as described. DNA ploidy was measured by propidium iodide staining using flow cytometric analysis over time. ( B ) Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv-Vpr at time 0 and then collected at 5 hours after viral transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser 345 antibody ( a ). The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as controls ( b ). ( C ). Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv or Adv-Vpr at time 0 and then collected at 11 hours after viral transduction for flow cytometric analysis. Ctr, control.

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser 345 phosphorylation and G2 arrest in HeLa cells . (A) a . Vpr induces cellular gross enlargement (top) with single enlarged nuclei (bottom). HeLa cells were synchronized in G1/S as described. Cells were then stained with DAPI. Images were captured 11 hours after Vpr transduction using a Leica DMR fluorescence microscope (DM4500B; Leica Microsystems) equipped with a high-performance camera (Hamamatsu) under visual light (top) and fluorescence (bottom). Scale bar: 10 μm. b . Vpr promotes the accumulation of DNA polyploidy as indicated by presence of 8N DNA. HeLa cells were synchronized in G1/S as described. DNA ploidy was measured by propidium iodide staining using flow cytometric analysis over time. ( B ) Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv-Vpr at time 0 and then collected at 5 hours after viral transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser 345 antibody ( a ). The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as controls ( b ). ( C ). Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv or Adv-Vpr at time 0 and then collected at 11 hours after viral transduction for flow cytometric analysis. Ctr, control.

    Article Snippet: Rabbit monoclonal anti-phospho-Chk1-Ser 345 (133D3) antibody was purchased from Cell Signaling Technology, Inc (Danvers, MA).

    Techniques: Staining, Transduction, Fluorescence, Microscopy, Western Blot

    Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser 345 phosphorylation and G2 arrest in CEM-SS cells . (A) Vpr promotes accumulation of DNA polyploidy as indicated by the presence of 8N DNA. Asynchronized CEM-SS cells were grown under the normal cell culture condition, and transduced with Adv viral control or Adv-Vpr. Cells were collected at indicated time point and DNA ploidy was measured by PI staining using flow cytometric analysis. ( B ) Asynchronized CEM-SS cells were pretreated with Cdt1, Cdc6 or control (Ctr) siRNA, and then transduced with Adv or Adv-Vpr 24 hours after addition of siRNAs. Cells were then harvested 48 hours post-transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser 345 antibody. The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as protein loading controls. ( C ). CEM-SS were treated the same way as described in ( B ). The cells were harvested 48 hours post-transduction and the cell lysates were then subjected to flow cytometric analysis.

    Journal: Retrovirology

    Article Title: Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

    doi: 10.1186/1742-4690-7-59

    Figure Lengend Snippet: Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser 345 phosphorylation and G2 arrest in CEM-SS cells . (A) Vpr promotes accumulation of DNA polyploidy as indicated by the presence of 8N DNA. Asynchronized CEM-SS cells were grown under the normal cell culture condition, and transduced with Adv viral control or Adv-Vpr. Cells were collected at indicated time point and DNA ploidy was measured by PI staining using flow cytometric analysis. ( B ) Asynchronized CEM-SS cells were pretreated with Cdt1, Cdc6 or control (Ctr) siRNA, and then transduced with Adv or Adv-Vpr 24 hours after addition of siRNAs. Cells were then harvested 48 hours post-transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser 345 antibody. The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as protein loading controls. ( C ). CEM-SS were treated the same way as described in ( B ). The cells were harvested 48 hours post-transduction and the cell lysates were then subjected to flow cytometric analysis.

    Article Snippet: Rabbit monoclonal anti-phospho-Chk1-Ser 345 (133D3) antibody was purchased from Cell Signaling Technology, Inc (Danvers, MA).

    Techniques: Cell Culture, Transduction, Staining, Western Blot

    PARP inhibition activates ATM. ( A ) Western blot for ATM phospho serine 1981 and ATM control following PARP inhibition for the times indicated. ( B ) Western blot for CHK1 phospho serine 345, CHK2 phospho threonine 68 and total CHK2 and actin controls following PARP inhibition or 0.5 mM HU treatment for 24 h.

    Journal: Nucleic Acids Research

    Article Title: Inhibition of poly (ADP-ribose) polymerase activates ATM which is required for subsequent homologous recombination repair

    doi: 10.1093/nar/gkl108

    Figure Lengend Snippet: PARP inhibition activates ATM. ( A ) Western blot for ATM phospho serine 1981 and ATM control following PARP inhibition for the times indicated. ( B ) Western blot for CHK1 phospho serine 345, CHK2 phospho threonine 68 and total CHK2 and actin controls following PARP inhibition or 0.5 mM HU treatment for 24 h.

    Article Snippet: This membrane was blocked in 5% milk for 1 h and immunoblotted with rabbit polyclonal antibodies against phospho-Ser 1981 ATM (Rockland 1:1000), total ATM (Oncogene 1:500), phospho-Ser 345 Chk1 (Cell Signaling 1:1000), phospho-Thr 68 Chk2 (Cell Signaling 1:1000), total Chk2 (Cell Signaling 1:1000) and β-actin (sigma 1:2000) proteins in 5% milk overnight.

    Techniques: Inhibition, Western Blot

    (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of pCHK1/CHK1 and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.

    Journal: PLoS ONE

    Article Title: Mutation of Serine 1333 in the ATR HEAT Repeats Creates a Hyperactive Kinase

    doi: 10.1371/journal.pone.0099397

    Figure Lengend Snippet: (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of pCHK1/CHK1 and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.

    Article Snippet: Antibodies used include ATR-N19 (Santa Cruz Biotechnology), HA (Covance), CHK1-G4 (Santa Cruz Biotechnology), FLAG-M2 (Sigma), ATRIP403 , MCM2 (BD Transduction Labs), phosphorylated Ser-317 CHK1 (Cell Signaling Technology), phosphorylated Ser-345 CHK1 (Cell Signaling Technology), and phosphorylated Ser-10 Histone H3 (Cell Signaling Technology).

    Techniques: Clone Assay, Expressing, SDS Page, Western Blot

    Real-time PCR analysis of RRM2 ( A , D ) gene expression and number of surviving cells ( B , E ) under RRM2 siRNA knockdown for NCI-H295R and MUC-1, respectively. RRM2 gene expression upon etoposide and doxorubicin treatment for NCI-H295R and MUC-1 ( C , F ). Quantification of Western blots for p-Chk1 ( G ), p-Chk2 ( H ), and p-H2AX ( I ) for no treatment, gemcitabine, cisplatin, and combination of both (gemcitabine and cisplatin) 24 h after treatment. Schematic illustration of the related DNA damage–repair pathway, including relevant therapeutic inhibitors ( J ). Stars represent significance vs. nontreated (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001); ns, non-significant.

    Journal: Cancers

    Article Title: Novel Insights into the Molecular Regulation of Ribonucleotide Reductase in Adrenocortical Carcinoma Treatment

    doi: 10.3390/cancers13164200

    Figure Lengend Snippet: Real-time PCR analysis of RRM2 ( A , D ) gene expression and number of surviving cells ( B , E ) under RRM2 siRNA knockdown for NCI-H295R and MUC-1, respectively. RRM2 gene expression upon etoposide and doxorubicin treatment for NCI-H295R and MUC-1 ( C , F ). Quantification of Western blots for p-Chk1 ( G ), p-Chk2 ( H ), and p-H2AX ( I ) for no treatment, gemcitabine, cisplatin, and combination of both (gemcitabine and cisplatin) 24 h after treatment. Schematic illustration of the related DNA damage–repair pathway, including relevant therapeutic inhibitors ( J ). Stars represent significance vs. nontreated (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001); ns, non-significant.

    Article Snippet: Antibodies used: RRM2 (#ab57653, Abcam), rabbit Ser 345 p-Chk1 (#2348, Cell Signaling, Danvers, MA, USA), rabbit Thr68 p-Chk2 (#2661, Cell Signaling), p-H2AX (#9718, Cell Signaling), and beta- actin (#A5316, Sigma-Aldrich).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot