chk1 ser 345 Search Results


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  • 86
    Cell Signaling Technology Inc pser 345 chk1
    Identification of a phosphorylation-sensitive protein complex consisting of AATF and MRLC3. (A) An oriented (pSer/pThr) phosphopeptide library, biased towards the basophilic phosphorylation motif of <t>Chk1/2</t> and MK2, was immobilized on streptavidin beads. The phospho φXRXXpT and non-phosphorylated φXRXXT peptide libraries were screened for interaction against in vitro translated, 35S-Met-labelled proteins. (B) Identification of MRLC3 as a non-phospho binder occurred in pool 16B11 and through progressive subdivision to a single clone. (C) Yeast two-hybrid screening revealed AATF as an interactor of MRLC3. We further characterized this interaction through co-immunoprecipitation (co-IP), performed in the presence or absence of 1 μM okadaic acid (OA). FLAG.MRLC3 was immunoprecipitated from HEK293T cells co-expressing V5.AATF. FLAG.GFP served as a control. Lane 3 shows an interaction of FLAG.MRLC3 with V5.AATF, which was abolished by OA-mediated Ser/Thr phosphatase inhibition 1 h prior to lysis (lane 4). (D) The MRLC3:AATF complex is sensitive to UV-C-induced DNA damage. FLAG.MRLC3 and V5.AATF-expressing HEK293T cells were UV-C irradiated (20 J/m2) 30 min prior to lysis and IP with anti-FLAG beads. FLAG.GFP served as a negative control. While V5.AATF co-precipitated with FLAG.MRLC3 in the absence of UV-C, the interaction was abrogated in the presence of DNA damage. (E) Reversal of the co-IP experiment is shown in (D). Anti-FLAG IP reveals AATF.FLAG:V5.MRLC3 complexes that display strong sensitivity to UV-C-induced DNA damage. FLAG.GFP served as a negative control. (F) Endogenous AATF:MRLC complexes display UV-C sensitivity. AATF was immunoprecipitated from HCT116 cells that were mock-treated or exposed to UV-C (20 J/m2) 30 min prior to lysis and IP. GFP IP served as a negative control (lanes 1 and 2). While substantial amounts of MRLC co-immunoprecipitated with AATF (lane 3), this interaction was abolished by UV-C-induced DNA damage (lane 4). (G) Endogenous AATF:MRLC3 complexes are sensitive to the topoisomerase-II inhibitor doxorubicin. AATF was immunoprecipitated from HCT116 cells that were mock-treated or exposed to doxorubicin (1 μM) 1 h prior to IP. GFP antibody served as a negative control (lanes 1 and 2). Doxorubicin (lane 4) disrupted the interaction between AATF and MRLC (lane 3). Figure source data can be found with the Supplementary data.
    Pser 345 Chk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Epitomics corp phospho 345 chk1
    WRN‐mediated NF‐κB activation requires <t>CHK1</t> and PARP1. (a) U2‐OS Cells were treated with CPT (50 nM) for indicated time periods, and activation of DDR proteins was assessed by Western blotting. (b) NEMO‐knockdown (NEMO‐KD) U2‐OS cells were generated by lentivirus mediated shRNA expression system. Expression of NEMO protein in NEMO‐WT (control shRNA) and NEMO‐KD cells was assessed by Western blotting. (c) NEMO‐WT and NEMO‐KD cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB activation was assessed in terms of IκBα degradation by Western blotting. (d) ATM‐knockdown (ATM‐KD) U2‐OS cells were generated by lentivirus mediated shRNA expression system. Expression of ATM protein in ATM‐WT (control shRNA) and ATM‐KD cells was assessed by Western blotting. (e) ATM‐WT and ATM‐KD cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB activation was assessed in term of IκBα degradation by Western blotting. (f) WRN‐WT cells expressing NF‐κB driven luciferase reporter were treated with CPT (50 nM) in the absence or presence of CHK1i or PARP1i for indicated time periods and NF‐κB activation was assessed in terms of fold increase in luciferase activity, which is normalized by renilla expression. All the values indicated are mean ± SD ( n = 3 for a, c, e) or mean ± SEM ( n = 4 for f). ** p < 0.01 with respect to vehicle treatment at respective time points
    Phospho 345 Chk1, supplied by Epitomics corp, 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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    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho 345 chk1 - by Bioz Stars, 2024-02
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    86
    Santa Cruz Biotechnology phospho chk1 345
    Erbb2 blocks <t>Chk1</t> activation through a PI3K/Akt dependent mechanism after UV irradiation. Keratinocytes in culture were treated with 45 μmol/L AG825 or the vehicle DMSO (A–D, F) or Erbb2fl/fl keratinocytes were infected with Cre recombinase expressing or empty virus (D) and sham-irradiated or exposed to 600 J/m2 UV 2 hours (for inhibitor) or 24 hours later (for Cre recombinase infection). Protein lysate was obtained at the indicated time points after UV irradiation (A–D, F). A: ATR/M activity was determined by immunoblotting for ATR/M substrate phosphorylation using an antibody specific for the phosphorylated consensus ATR/M substrate sequence and immunoblotting for actin. The sum of the signal from all bands detected with the ATR/M substrate phosphorylation antibody was normalized to actin levels using densitometry. The mean and SE for at least six samples at each time point and treatment is shown. No significant differences were detected between DMSO and AG825 treated samples at any time point. B: Chk1 phosphorylation on Ser345 was determined after immunoblotting with a phospho-Chk1-Ser345 specific antibody (inset). The signal for Chk1 phosphorylation relative to actin was determined using densitometry and the results from two replicate experiments averaged and graphed. Mean ± SEM is shown. C: Immunoblotting for Chk1 and Chk2 phosphorylation on abrogation of Erbb2 and UV exposure in cell lysates (“P” = phospho). D: Akt activation, measured by Akt phosphorylation (P-AKT), is dependent on Erbb2 activation after UV irradiation. “m” indicates minutes post-UV. E: Inhibition of PI3K or Akt, as described in Materials and Methods, causes S-phase arrest 24 hours after UV irradiation. N ≥4 experiments. Significantly different when compared with the vehicle-treated and UV-irradiated control, where *P ≤ 0.05. F: Immunoblotting for inhibitory phosphorylation of Chk1 on Ser280 on abrogation of Erbb2 activity and UV irradiation.
    Phospho Chk1 345, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho chk1 345/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho chk1 345 - by Bioz Stars, 2024-02
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    86
    Cell Signaling Technology Inc phosphoserine 345 chk1
    Erbb2 blocks <t>Chk1</t> activation through a PI3K/Akt dependent mechanism after UV irradiation. Keratinocytes in culture were treated with 45 μmol/L AG825 or the vehicle DMSO (A–D, F) or Erbb2fl/fl keratinocytes were infected with Cre recombinase expressing or empty virus (D) and sham-irradiated or exposed to 600 J/m2 UV 2 hours (for inhibitor) or 24 hours later (for Cre recombinase infection). Protein lysate was obtained at the indicated time points after UV irradiation (A–D, F). A: ATR/M activity was determined by immunoblotting for ATR/M substrate phosphorylation using an antibody specific for the phosphorylated consensus ATR/M substrate sequence and immunoblotting for actin. The sum of the signal from all bands detected with the ATR/M substrate phosphorylation antibody was normalized to actin levels using densitometry. The mean and SE for at least six samples at each time point and treatment is shown. No significant differences were detected between DMSO and AG825 treated samples at any time point. B: Chk1 phosphorylation on Ser345 was determined after immunoblotting with a phospho-Chk1-Ser345 specific antibody (inset). The signal for Chk1 phosphorylation relative to actin was determined using densitometry and the results from two replicate experiments averaged and graphed. Mean ± SEM is shown. C: Immunoblotting for Chk1 and Chk2 phosphorylation on abrogation of Erbb2 and UV exposure in cell lysates (“P” = phospho). D: Akt activation, measured by Akt phosphorylation (P-AKT), is dependent on Erbb2 activation after UV irradiation. “m” indicates minutes post-UV. E: Inhibition of PI3K or Akt, as described in Materials and Methods, causes S-phase arrest 24 hours after UV irradiation. N ≥4 experiments. Significantly different when compared with the vehicle-treated and UV-irradiated control, where *P ≤ 0.05. F: Immunoblotting for inhibitory phosphorylation of Chk1 on Ser280 on abrogation of Erbb2 activity and UV irradiation.
    Phosphoserine 345 Chk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phosphoserine 345 chk1/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phosphoserine 345 chk1 - by Bioz Stars, 2024-02
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    Image Search Results


    Identification of a phosphorylation-sensitive protein complex consisting of AATF and MRLC3. (A) An oriented (pSer/pThr) phosphopeptide library, biased towards the basophilic phosphorylation motif of Chk1/2 and MK2, was immobilized on streptavidin beads. The phospho φXRXXpT and non-phosphorylated φXRXXT peptide libraries were screened for interaction against in vitro translated, 35S-Met-labelled proteins. (B) Identification of MRLC3 as a non-phospho binder occurred in pool 16B11 and through progressive subdivision to a single clone. (C) Yeast two-hybrid screening revealed AATF as an interactor of MRLC3. We further characterized this interaction through co-immunoprecipitation (co-IP), performed in the presence or absence of 1 μM okadaic acid (OA). FLAG.MRLC3 was immunoprecipitated from HEK293T cells co-expressing V5.AATF. FLAG.GFP served as a control. Lane 3 shows an interaction of FLAG.MRLC3 with V5.AATF, which was abolished by OA-mediated Ser/Thr phosphatase inhibition 1 h prior to lysis (lane 4). (D) The MRLC3:AATF complex is sensitive to UV-C-induced DNA damage. FLAG.MRLC3 and V5.AATF-expressing HEK293T cells were UV-C irradiated (20 J/m2) 30 min prior to lysis and IP with anti-FLAG beads. FLAG.GFP served as a negative control. While V5.AATF co-precipitated with FLAG.MRLC3 in the absence of UV-C, the interaction was abrogated in the presence of DNA damage. (E) Reversal of the co-IP experiment is shown in (D). Anti-FLAG IP reveals AATF.FLAG:V5.MRLC3 complexes that display strong sensitivity to UV-C-induced DNA damage. FLAG.GFP served as a negative control. (F) Endogenous AATF:MRLC complexes display UV-C sensitivity. AATF was immunoprecipitated from HCT116 cells that were mock-treated or exposed to UV-C (20 J/m2) 30 min prior to lysis and IP. GFP IP served as a negative control (lanes 1 and 2). While substantial amounts of MRLC co-immunoprecipitated with AATF (lane 3), this interaction was abolished by UV-C-induced DNA damage (lane 4). (G) Endogenous AATF:MRLC3 complexes are sensitive to the topoisomerase-II inhibitor doxorubicin. AATF was immunoprecipitated from HCT116 cells that were mock-treated or exposed to doxorubicin (1 μM) 1 h prior to IP. GFP antibody served as a negative control (lanes 1 and 2). Doxorubicin (lane 4) disrupted the interaction between AATF and MRLC (lane 3). Figure source data can be found with the Supplementary data.

    Journal: The EMBO Journal

    Article Title: AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis

    doi: 10.1038/emboj.2012.236

    Figure Lengend Snippet: Identification of a phosphorylation-sensitive protein complex consisting of AATF and MRLC3. (A) An oriented (pSer/pThr) phosphopeptide library, biased towards the basophilic phosphorylation motif of Chk1/2 and MK2, was immobilized on streptavidin beads. The phospho φXRXXpT and non-phosphorylated φXRXXT peptide libraries were screened for interaction against in vitro translated, 35S-Met-labelled proteins. (B) Identification of MRLC3 as a non-phospho binder occurred in pool 16B11 and through progressive subdivision to a single clone. (C) Yeast two-hybrid screening revealed AATF as an interactor of MRLC3. We further characterized this interaction through co-immunoprecipitation (co-IP), performed in the presence or absence of 1 μM okadaic acid (OA). FLAG.MRLC3 was immunoprecipitated from HEK293T cells co-expressing V5.AATF. FLAG.GFP served as a control. Lane 3 shows an interaction of FLAG.MRLC3 with V5.AATF, which was abolished by OA-mediated Ser/Thr phosphatase inhibition 1 h prior to lysis (lane 4). (D) The MRLC3:AATF complex is sensitive to UV-C-induced DNA damage. FLAG.MRLC3 and V5.AATF-expressing HEK293T cells were UV-C irradiated (20 J/m2) 30 min prior to lysis and IP with anti-FLAG beads. FLAG.GFP served as a negative control. While V5.AATF co-precipitated with FLAG.MRLC3 in the absence of UV-C, the interaction was abrogated in the presence of DNA damage. (E) Reversal of the co-IP experiment is shown in (D). Anti-FLAG IP reveals AATF.FLAG:V5.MRLC3 complexes that display strong sensitivity to UV-C-induced DNA damage. FLAG.GFP served as a negative control. (F) Endogenous AATF:MRLC complexes display UV-C sensitivity. AATF was immunoprecipitated from HCT116 cells that were mock-treated or exposed to UV-C (20 J/m2) 30 min prior to lysis and IP. GFP IP served as a negative control (lanes 1 and 2). While substantial amounts of MRLC co-immunoprecipitated with AATF (lane 3), this interaction was abolished by UV-C-induced DNA damage (lane 4). (G) Endogenous AATF:MRLC3 complexes are sensitive to the topoisomerase-II inhibitor doxorubicin. AATF was immunoprecipitated from HCT116 cells that were mock-treated or exposed to doxorubicin (1 μM) 1 h prior to IP. GFP antibody served as a negative control (lanes 1 and 2). Doxorubicin (lane 4) disrupted the interaction between AATF and MRLC (lane 3). Figure source data can be found with the Supplementary data.

    Article Snippet: St. Hybridoma Bank), Chk1, pSer-345 Chk1, MK2, pThr-334 MK2, Puma, Bax, p53, Gadd45α (Cell Signalling), MRLC (Santa Cruz and homemade), p21 (Santa Cruz), p53 (Dako) were used for immunoprecipitation, immunoblotting, immunofluorescence immunohistochemistry and FACS.

    Techniques: In Vitro, Two Hybrid Screening, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Inhibition, Lysis, Irradiation, Negative Control

    WRN‐mediated NF‐κB activation requires CHK1 and PARP1. (a) U2‐OS Cells were treated with CPT (50 nM) for indicated time periods, and activation of DDR proteins was assessed by Western blotting. (b) NEMO‐knockdown (NEMO‐KD) U2‐OS cells were generated by lentivirus mediated shRNA expression system. Expression of NEMO protein in NEMO‐WT (control shRNA) and NEMO‐KD cells was assessed by Western blotting. (c) NEMO‐WT and NEMO‐KD cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB activation was assessed in terms of IκBα degradation by Western blotting. (d) ATM‐knockdown (ATM‐KD) U2‐OS cells were generated by lentivirus mediated shRNA expression system. Expression of ATM protein in ATM‐WT (control shRNA) and ATM‐KD cells was assessed by Western blotting. (e) ATM‐WT and ATM‐KD cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB activation was assessed in term of IκBα degradation by Western blotting. (f) WRN‐WT cells expressing NF‐κB driven luciferase reporter were treated with CPT (50 nM) in the absence or presence of CHK1i or PARP1i for indicated time periods and NF‐κB activation was assessed in terms of fold increase in luciferase activity, which is normalized by renilla expression. All the values indicated are mean ± SD ( n = 3 for a, c, e) or mean ± SEM ( n = 4 for f). ** p < 0.01 with respect to vehicle treatment at respective time points

    Journal: Aging Cell

    Article Title: Non‐enzymatic function of WRN RECQL helicase regulates removal of topoisomerase‐I‐DNA covalent complexes and triggers NF‐κB signaling in cancer

    doi: 10.1111/acel.13625

    Figure Lengend Snippet: WRN‐mediated NF‐κB activation requires CHK1 and PARP1. (a) U2‐OS Cells were treated with CPT (50 nM) for indicated time periods, and activation of DDR proteins was assessed by Western blotting. (b) NEMO‐knockdown (NEMO‐KD) U2‐OS cells were generated by lentivirus mediated shRNA expression system. Expression of NEMO protein in NEMO‐WT (control shRNA) and NEMO‐KD cells was assessed by Western blotting. (c) NEMO‐WT and NEMO‐KD cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB activation was assessed in terms of IκBα degradation by Western blotting. (d) ATM‐knockdown (ATM‐KD) U2‐OS cells were generated by lentivirus mediated shRNA expression system. Expression of ATM protein in ATM‐WT (control shRNA) and ATM‐KD cells was assessed by Western blotting. (e) ATM‐WT and ATM‐KD cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB activation was assessed in term of IκBα degradation by Western blotting. (f) WRN‐WT cells expressing NF‐κB driven luciferase reporter were treated with CPT (50 nM) in the absence or presence of CHK1i or PARP1i for indicated time periods and NF‐κB activation was assessed in terms of fold increase in luciferase activity, which is normalized by renilla expression. All the values indicated are mean ± SD ( n = 3 for a, c, e) or mean ± SEM ( n = 4 for f). ** p < 0.01 with respect to vehicle treatment at respective time points

    Article Snippet: Anti‐phospho‐345‐CHK1 was from Epitomics (Cambridge, UK).

    Techniques: Activation Assay, Western Blot, Generated, shRNA, Expressing, Luciferase, Activity Assay

    Non‐enzymatic role of WRN in ssDNA generation and activation of CHK1 and NF‐κB. (a) WRN‐KO cells were transfected for ectopic expression of EV (empty vector), WRN WT , WRN E84A , WRN K577M , and WRN E84A‐K577M . Cells were treated with CPT (50 nM) for indicated time periods, and phosphorylation of RPA2 and CHK1 was assessed by Western blotting. (b) WRN‐KO cells were transfected for ectopic expression of EV (empty vector), WRN WT , WRN E84A , WRN K577M , and WRN E84A‐K577M . Cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB promoter driven luciferase expression was assessed. All the values indicated are mean ± SEM ( n = 4). * p < 0.05 with respect to respective cell types at 0 h

    Journal: Aging Cell

    Article Title: Non‐enzymatic function of WRN RECQL helicase regulates removal of topoisomerase‐I‐DNA covalent complexes and triggers NF‐κB signaling in cancer

    doi: 10.1111/acel.13625

    Figure Lengend Snippet: Non‐enzymatic role of WRN in ssDNA generation and activation of CHK1 and NF‐κB. (a) WRN‐KO cells were transfected for ectopic expression of EV (empty vector), WRN WT , WRN E84A , WRN K577M , and WRN E84A‐K577M . Cells were treated with CPT (50 nM) for indicated time periods, and phosphorylation of RPA2 and CHK1 was assessed by Western blotting. (b) WRN‐KO cells were transfected for ectopic expression of EV (empty vector), WRN WT , WRN E84A , WRN K577M , and WRN E84A‐K577M . Cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB promoter driven luciferase expression was assessed. All the values indicated are mean ± SEM ( n = 4). * p < 0.05 with respect to respective cell types at 0 h

    Article Snippet: Anti‐phospho‐345‐CHK1 was from Epitomics (Cambridge, UK).

    Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Luciferase

    Role of WRN in ssDNA generation and nuclear translocation of p65 and its therapeutic implications. (a) Schematic representation for WRN‐mediated removal of TOP1cc, generation of ssDNA and CHK1 phosphorylation and NF‐κB activation. (b, c) WRN‐WT and WRN‐KO cells were treated with BrdU for 36 h for uniform labeling of BrdU on both the DNA strands. Further cells were exposed to CPT for indicated time periods for TOP1cc removal mediated ssDNA generation and nuclear translocation of NF‐κB (p65). BrdU exposed in ssDNA (red) and p65 (green) was assessed in non‐denaturing native condition by immunofluorescence microscopy (Bar: 5 μm). % Nuclei with more than threshold fluorescence were considered positive for p65 and/or BrdU and plotted in C. All the values indicated are mean ± SEM ( n = 3). * p < 0.05 with respect to respective untreated cell types. (d,e) Mice bearing WRN‐WT and WRN‐KO melanoma tumors were treated with vehicle or CPT (1 mg/kg, once on 1st, 3rd, and 5th day per week for 4 weeks). Tumor volume was measured once every alternate day. After 30 days, mice were sacrificed and tumors were removed and analyzed. Data represent the mean ± SD , n = 6 per group. * p < 0.01 w.r.t corresponding vehicle‐treated tumor. (f) Survival of patients with WRN high and low expression in cancer using data available at cBioportal. The EXP < −1 denotes mRNA expression is <1 standard deviations ( SD ) below the mean, and EXP > 1 denotes mRNA expression is >1 SD above the mean. (g) Schematic model for WRN‐mediated TOP1cc removal and NF‐κB activation. In response to TOP1cc, WRN plays key role in removal of TOP1cc, leading to generation of RPA coated ssDNA and activation of CHK1 and PARP1. Subsequently, CHK1 and PARP1 may facilitate NEMO translocation to cytoplasm and activation of NF‐κB. [Correction added on 01 June 2022, after first online publication: Figure labels, 6(e) and 6(f) are misplaced and have been corrected in this version].

    Journal: Aging Cell

    Article Title: Non‐enzymatic function of WRN RECQL helicase regulates removal of topoisomerase‐I‐DNA covalent complexes and triggers NF‐κB signaling in cancer

    doi: 10.1111/acel.13625

    Figure Lengend Snippet: Role of WRN in ssDNA generation and nuclear translocation of p65 and its therapeutic implications. (a) Schematic representation for WRN‐mediated removal of TOP1cc, generation of ssDNA and CHK1 phosphorylation and NF‐κB activation. (b, c) WRN‐WT and WRN‐KO cells were treated with BrdU for 36 h for uniform labeling of BrdU on both the DNA strands. Further cells were exposed to CPT for indicated time periods for TOP1cc removal mediated ssDNA generation and nuclear translocation of NF‐κB (p65). BrdU exposed in ssDNA (red) and p65 (green) was assessed in non‐denaturing native condition by immunofluorescence microscopy (Bar: 5 μm). % Nuclei with more than threshold fluorescence were considered positive for p65 and/or BrdU and plotted in C. All the values indicated are mean ± SEM ( n = 3). * p < 0.05 with respect to respective untreated cell types. (d,e) Mice bearing WRN‐WT and WRN‐KO melanoma tumors were treated with vehicle or CPT (1 mg/kg, once on 1st, 3rd, and 5th day per week for 4 weeks). Tumor volume was measured once every alternate day. After 30 days, mice were sacrificed and tumors were removed and analyzed. Data represent the mean ± SD , n = 6 per group. * p < 0.01 w.r.t corresponding vehicle‐treated tumor. (f) Survival of patients with WRN high and low expression in cancer using data available at cBioportal. The EXP < −1 denotes mRNA expression is <1 standard deviations ( SD ) below the mean, and EXP > 1 denotes mRNA expression is >1 SD above the mean. (g) Schematic model for WRN‐mediated TOP1cc removal and NF‐κB activation. In response to TOP1cc, WRN plays key role in removal of TOP1cc, leading to generation of RPA coated ssDNA and activation of CHK1 and PARP1. Subsequently, CHK1 and PARP1 may facilitate NEMO translocation to cytoplasm and activation of NF‐κB. [Correction added on 01 June 2022, after first online publication: Figure labels, 6(e) and 6(f) are misplaced and have been corrected in this version].

    Article Snippet: Anti‐phospho‐345‐CHK1 was from Epitomics (Cambridge, UK).

    Techniques: Translocation Assay, Activation Assay, Labeling, Immunofluorescence, Microscopy, Fluorescence, Expressing

    Erbb2 blocks Chk1 activation through a PI3K/Akt dependent mechanism after UV irradiation. Keratinocytes in culture were treated with 45 μmol/L AG825 or the vehicle DMSO (A–D, F) or Erbb2fl/fl keratinocytes were infected with Cre recombinase expressing or empty virus (D) and sham-irradiated or exposed to 600 J/m2 UV 2 hours (for inhibitor) or 24 hours later (for Cre recombinase infection). Protein lysate was obtained at the indicated time points after UV irradiation (A–D, F). A: ATR/M activity was determined by immunoblotting for ATR/M substrate phosphorylation using an antibody specific for the phosphorylated consensus ATR/M substrate sequence and immunoblotting for actin. The sum of the signal from all bands detected with the ATR/M substrate phosphorylation antibody was normalized to actin levels using densitometry. The mean and SE for at least six samples at each time point and treatment is shown. No significant differences were detected between DMSO and AG825 treated samples at any time point. B: Chk1 phosphorylation on Ser345 was determined after immunoblotting with a phospho-Chk1-Ser345 specific antibody (inset). The signal for Chk1 phosphorylation relative to actin was determined using densitometry and the results from two replicate experiments averaged and graphed. Mean ± SEM is shown. C: Immunoblotting for Chk1 and Chk2 phosphorylation on abrogation of Erbb2 and UV exposure in cell lysates (“P” = phospho). D: Akt activation, measured by Akt phosphorylation (P-AKT), is dependent on Erbb2 activation after UV irradiation. “m” indicates minutes post-UV. E: Inhibition of PI3K or Akt, as described in Materials and Methods, causes S-phase arrest 24 hours after UV irradiation. N ≥4 experiments. Significantly different when compared with the vehicle-treated and UV-irradiated control, where *P ≤ 0.05. F: Immunoblotting for inhibitory phosphorylation of Chk1 on Ser280 on abrogation of Erbb2 activity and UV irradiation.

    Journal:

    Article Title: Erbb2 Suppresses DNA Damage-Induced Checkpoint Activation and UV-Induced Mouse Skin Tumorigenesis

    doi: 10.2353/ajpath.2009.080638

    Figure Lengend Snippet: Erbb2 blocks Chk1 activation through a PI3K/Akt dependent mechanism after UV irradiation. Keratinocytes in culture were treated with 45 μmol/L AG825 or the vehicle DMSO (A–D, F) or Erbb2fl/fl keratinocytes were infected with Cre recombinase expressing or empty virus (D) and sham-irradiated or exposed to 600 J/m2 UV 2 hours (for inhibitor) or 24 hours later (for Cre recombinase infection). Protein lysate was obtained at the indicated time points after UV irradiation (A–D, F). A: ATR/M activity was determined by immunoblotting for ATR/M substrate phosphorylation using an antibody specific for the phosphorylated consensus ATR/M substrate sequence and immunoblotting for actin. The sum of the signal from all bands detected with the ATR/M substrate phosphorylation antibody was normalized to actin levels using densitometry. The mean and SE for at least six samples at each time point and treatment is shown. No significant differences were detected between DMSO and AG825 treated samples at any time point. B: Chk1 phosphorylation on Ser345 was determined after immunoblotting with a phospho-Chk1-Ser345 specific antibody (inset). The signal for Chk1 phosphorylation relative to actin was determined using densitometry and the results from two replicate experiments averaged and graphed. Mean ± SEM is shown. C: Immunoblotting for Chk1 and Chk2 phosphorylation on abrogation of Erbb2 and UV exposure in cell lysates (“P” = phospho). D: Akt activation, measured by Akt phosphorylation (P-AKT), is dependent on Erbb2 activation after UV irradiation. “m” indicates minutes post-UV. E: Inhibition of PI3K or Akt, as described in Materials and Methods, causes S-phase arrest 24 hours after UV irradiation. N ≥4 experiments. Significantly different when compared with the vehicle-treated and UV-irradiated control, where *P ≤ 0.05. F: Immunoblotting for inhibitory phosphorylation of Chk1 on Ser280 on abrogation of Erbb2 activity and UV irradiation.

    Article Snippet: Membranes were immunoblotted with antibodies recognizing actin (Sigma), phosphorylated ATR/M substrates (Cell Signaling, Danvers, MA), Cdc25a (Santa Cruz Biotechnology, Santa Cruz, CA), Cdc25b (Cell Signaling), Cdc25c (Santa Cruz Biotechnology), phospho-Chk1-Ser 296 (Cell Signaling), phospho-Chk1-Ser 280 (gift of Ramon Parsons), phospho-Chk1 345 (Santa Cruz), phospho-Chk2 387 (Cell Signaling), and phospho-Akt (Cell Signaling, Beverly, MA), horseradish peroxidase-conjugated secondary antibodies (Cell Signaling), and visualized using chemiluminescent reagents (Pierce, Rockford, IL).

    Techniques: Activation Assay, Irradiation, Infection, Expressing, Activity Assay, Western Blot, Sequencing, Inhibition

    Erbb2 regulates cell cycle progression following UV exposure by suppressing ATR checkpoint activation. A: In normal cells, UV-induced Erbb2 activation in turn activates PI3K/Akt signaling, which suppresses Chk1 activation and subsequent degradation of Cdc25a. B: On abrogation of Erbb2 activity, increased Chk1/2 activity leads to decreased Cdc25a and subsequently, to increased S-phase arrest. The net effect of decreased Erbb2 activity is decreased skin tumor development, presumably because of improved DNA repair and decreased mutagenesis.

    Journal:

    Article Title: Erbb2 Suppresses DNA Damage-Induced Checkpoint Activation and UV-Induced Mouse Skin Tumorigenesis

    doi: 10.2353/ajpath.2009.080638

    Figure Lengend Snippet: Erbb2 regulates cell cycle progression following UV exposure by suppressing ATR checkpoint activation. A: In normal cells, UV-induced Erbb2 activation in turn activates PI3K/Akt signaling, which suppresses Chk1 activation and subsequent degradation of Cdc25a. B: On abrogation of Erbb2 activity, increased Chk1/2 activity leads to decreased Cdc25a and subsequently, to increased S-phase arrest. The net effect of decreased Erbb2 activity is decreased skin tumor development, presumably because of improved DNA repair and decreased mutagenesis.

    Article Snippet: Membranes were immunoblotted with antibodies recognizing actin (Sigma), phosphorylated ATR/M substrates (Cell Signaling, Danvers, MA), Cdc25a (Santa Cruz Biotechnology, Santa Cruz, CA), Cdc25b (Cell Signaling), Cdc25c (Santa Cruz Biotechnology), phospho-Chk1-Ser 296 (Cell Signaling), phospho-Chk1-Ser 280 (gift of Ramon Parsons), phospho-Chk1 345 (Santa Cruz), phospho-Chk2 387 (Cell Signaling), and phospho-Akt (Cell Signaling, Beverly, MA), horseradish peroxidase-conjugated secondary antibodies (Cell Signaling), and visualized using chemiluminescent reagents (Pierce, Rockford, IL).

    Techniques: Activation Assay, Activity Assay, Mutagenesis