Journal: eLife

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

doi: 10.7554/eLife.86976

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

Article Snippet: Other antibodies include α-tubulin (Santa Cruz Biotechnology, #sc-5286), E6AP (Bethyl Laboratories, A300-351), HA (Cell Signaling Technology #3724), γ-H2AX Ser-139 (Cell Signaling Technology #9718S), phospho-ATM/ATR substrate motif (Cell Signaling Technology, #6966S), phospho-SMC1 Ser-957 (Cell Signaling Technology, #58052), phospho-CHK1 Ser-345 (Cell Signaling Technology, #2348), phospho-CHK2 Thr-68 (Cell Signaling Technology, #2197), Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (Cell Signaling Technology, #2914), Aurora A (Cell Signaling Technology, #14475), Aurora B (Cell Signaling Technology, #3094), CDK1 (Cell Signaling Technology, #9112), Cyclin B (Cell Signaling Technology, #4138), phosphor-CDK substrates (Cell Signaling Technology, #2325), RPA32 (Thermo Fisher Scientific, #PA5-22256), S5a (Boston Biochem, #SP-400), ubiquitin (Cell Signaling Technology, #3936), and GFP (Cell Signaling Technology, #2555).

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

Journal: eLife

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

doi: 10.7554/eLife.86976

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

Article Snippet: Other antibodies include α-tubulin (Santa Cruz Biotechnology, #sc-5286), E6AP (Bethyl Laboratories, A300-351), HA (Cell Signaling Technology #3724), γ-H2AX Ser-139 (Cell Signaling Technology #9718S), phospho-ATM/ATR substrate motif (Cell Signaling Technology, #6966S), phospho-SMC1 Ser-957 (Cell Signaling Technology, #58052), phospho-CHK1 Ser-345 (Cell Signaling Technology, #2348), phospho-CHK2 Thr-68 (Cell Signaling Technology, #2197), Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (Cell Signaling Technology, #2914), Aurora A (Cell Signaling Technology, #14475), Aurora B (Cell Signaling Technology, #3094), CDK1 (Cell Signaling Technology, #9112), Cyclin B (Cell Signaling Technology, #4138), phosphor-CDK substrates (Cell Signaling Technology, #2325), RPA32 (Thermo Fisher Scientific, #PA5-22256), S5a (Boston Biochem, #SP-400), ubiquitin (Cell Signaling Technology, #3936), and GFP (Cell Signaling Technology, #2555).

Techniques: Expressing, Immunoprecipitation, Western Blot

Journal: iScience

Article Title: TZAP overexpression induces telomere dysfunction and ALT-like activity in ATRX/DAXX-deficient cells

doi: 10.1016/j.isci.2023.106405

Figure Lengend Snippet: Key resources table

Article Snippet: . α-GAPDH (# PAB17013 , Abnova), α-γH2A.x Ser 139 (JBW301, #05-636, Millipore), α-Myc-tag (9B11, #2276S, Cell Signaling), α-p-Chk1 Ser 345 (133D3, #2348S, Cell Signaling), α-PML (#sc-5621, Santa Cruz) α-p-Chk2 Thr 68 (#2661S, Cell Signaling), α-p-ATR Thr 1989 (#ab223258, Abcam), α-p-ATM Ser 1981 (D25E5, #13050S, Cell Signaling), α-ATRX (#sc-15408, Santa Cruz), α-DAXX (#sc-7152, Santa Cruz), α-H3.3 (#ab176840, Abcam), α-RMI1 (#NB100-1720, Novus), α-FLAG-tag (M2, #F1804-1MG, Sigma), α-RPA32 (9H8, #ab2175, Abcam), α-RPA70 (#sc-28304, Santa Cruz). α-BLM (R7019), α-TRF1 (R6839) and α-TRF2 (R6841) were raised in our lab. All antibodies were used for Western Blotting apart from α-BLM, α-Myc and α-FLAG that were also used for IF and α-RPA32, α-RPA70, α-PML, α-TRF1 and α-TRF2 that were used for IF only.

Techniques: Recombinant, Modification, Transfection, Electrophoresis, Blocking Assay, Imaging, Purification, Software

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

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

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

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

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

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:

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

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