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Addgene inc ube3a insert
Ube3a Insert, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/p4054+e6ap/pm39798888-65-13-16?v=Addgene+inc
Average 91 stars, based on 9 article reviews

ube3a insert - by Bioz Stars, 2026-08

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( A ) Immunoprecipitation (IP) was performed in HeLa cell lysates, as described in Materials and methods. The lysate input, MASTL IP, and control (ctr) IP samples were analyzed by immunoblotting for E6AP and MASTL. ( B ) A pulldown assay was performed in HeLa cell lysates using MBP-tagged E6AP, as described in Materials and methods. The pulldown product, cell lysis input, and a control (-) pulldown (using empty beads) were analyzed by immunoblotting for E6AP and MASTL. ( C ) A pulldown assay was performed using MBP-tagged full-length E6AP in Xenopus egg extract. Purified segments of MASTL, including N (aa 1–340), M (aa 335–660), and C (aa 656–887), were supplemented in the extracts. The pulldown products, egg extract inputs, and a control (-) pulldown (using empty beads) were analyzed by immunoblotting for GST and MBP. ( D ) Segments of E6AP, including N (aa 1–280), M (aa 280–497), and C (aa 497–770), were tagged with GFP, and transfected into HeLa cells for expression. 24 hr after transfection, cell lysates were harvested for GFP IP. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for MASTL and GFP.

Journal: eLife

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

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) Immunoprecipitation (IP) was performed in HeLa cell lysates, as described in Materials and methods. The lysate input, MASTL IP, and control (ctr) IP samples were analyzed by immunoblotting for E6AP and MASTL. ( B ) A pulldown assay was performed in HeLa cell lysates using MBP-tagged E6AP, as described in Materials and methods. The pulldown product, cell lysis input, and a control (-) pulldown (using empty beads) were analyzed by immunoblotting for E6AP and MASTL. ( C ) A pulldown assay was performed using MBP-tagged full-length E6AP in Xenopus egg extract. Purified segments of MASTL, including N (aa 1–340), M (aa 335–660), and C (aa 656–887), were supplemented in the extracts. The pulldown products, egg extract inputs, and a control (-) pulldown (using empty beads) were analyzed by immunoblotting for GST and MBP. ( D ) Segments of E6AP, including N (aa 1–280), M (aa 280–497), and C (aa 497–770), were tagged with GFP, and transfected into HeLa cells for expression. 24 hr after transfection, cell lysates were harvested for GFP IP. The input, GFP IP, and control (ctr) IP using blank beads were analyzed by immunoblotting for MASTL and GFP.

Article Snippet: A vector expressing HA-tagged human E6AP was obtained from Addgene (Plasmid #8658), E6AP mutants were generated using site-directed mutagenesis (Agilent) following the protocol recommended by the manufacturer.

Techniques: Immunoprecipitation, Control, Western Blot, Lysis, Purification, Transfection, Expressing

( A ) MBP-E6AP and GST-MASTL were expressed and purified, as described in Materials and methods. GST-MASTL on glutathione beads, or control glutathione beads, were incubated with MBP-E6AP, followed by pulldown, as described in Materials and methods. The input, GST-MASTL pulldown, and control pull down samples were analyzed by immunoblotting for MASTL and E6AP. ( B ) GFP-MASTL, FL or N-terminus (aa 1–340), was expressed in HeLa cells. Immunoprecipitation (IP) was performed using a GFP antibody, and the presence of E6AP in the IP products was examined by immunoblotting. ( C ) IP was performed using GFP-tagged segments of E6AP expressed in HeLa cells. The IP product, cell lysate input, and a control IP (using empty beads) were analyzed by immunoblotting for MASTL and GFP. N: aa 1–280; N1: aa 1–99; N2: aa 100–207; N3: aa 108–280.

Journal: eLife

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

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) MBP-E6AP and GST-MASTL were expressed and purified, as described in Materials and methods. GST-MASTL on glutathione beads, or control glutathione beads, were incubated with MBP-E6AP, followed by pulldown, as described in Materials and methods. The input, GST-MASTL pulldown, and control pull down samples were analyzed by immunoblotting for MASTL and E6AP. ( B ) GFP-MASTL, FL or N-terminus (aa 1–340), was expressed in HeLa cells. Immunoprecipitation (IP) was performed using a GFP antibody, and the presence of E6AP in the IP products was examined by immunoblotting. ( C ) IP was performed using GFP-tagged segments of E6AP expressed in HeLa cells. The IP product, cell lysate input, and a control IP (using empty beads) were analyzed by immunoblotting for MASTL and GFP. N: aa 1–280; N1: aa 1–99; N2: aa 100–207; N3: aa 108–280.

Techniques: Purification, Control, Incubation, Western Blot, Immunoprecipitation

( A ) HeLa cells were transfected with control or E6AP-targeting siRNA E6AP for 24 hr. Cells were analyzed by immunoblotting for E6AP, MASTL, and β-actin. ( B ) HeLa cells were transfected with HA-tagged E6AP. 24 hr after transfection, cells were analyzed by immunoblotting for E6AP, MASLT, and α-tubulin. ( C ) As in panel B, HeLa cells were transfected with HA-E6AP, and analyzed by immunofluorescence (IF) for HA (green) and MASTL (red). Cells with HA-E6AP expression, as denoted by arrowheads, exhibited lower MASTL expression. ( D ) E6AP gene knockout (KO) was performed in HeLa cells, as described in Materials and methods. HA-E6AP was expressed in E6AP KO cells, as indicated. Cells were analyzed by immunoblotting for MASTL, E6AP, and α-tubulin. ( E ) HeLa cells transfected with control or E6AP siRNA were treated with 20 μg/ml cycloheximide (CHX), as indicated. Cells were harvested and analyzed by immunoblotting for MASTL and β-actin. ( F ) HeLa cells were treated as in panel E, MASTL and β-actin protein levels were quantified, and the ratio is shown for the indicated time points after CHX treatment, after normalized to that of time 0. The mean values and standard deviations were calculated from three experiments. ( G ) WT or E6AP KO HeLa cells were transfected with HA-tagged ubiquitin for 12 hr, followed 50 μM MG132 treatment for 4 hr. Cell lysates were harvested for HA immunoprecipitation (IP) or ctr IP using blank beads. The input and IP products were analyzed by immunoblotting for MASTL and HA. ( H ) In vitro ubiquitination assay was performed using E6AP as E3 ligase, and MASTL as substrate, as described in Materials and methods. S5a was added as a control substrate. The reactions were incubated as indicated, as analyzed by immunoblotting for MASTL, ubiquitination, and S5a.

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 were transfected with control or E6AP-targeting siRNA E6AP for 24 hr. Cells were analyzed by immunoblotting for E6AP, MASTL, and β-actin. ( B ) HeLa cells were transfected with HA-tagged E6AP. 24 hr after transfection, cells were analyzed by immunoblotting for E6AP, MASLT, and α-tubulin. ( C ) As in panel B, HeLa cells were transfected with HA-E6AP, and analyzed by immunofluorescence (IF) for HA (green) and MASTL (red). Cells with HA-E6AP expression, as denoted by arrowheads, exhibited lower MASTL expression. ( D ) E6AP gene knockout (KO) was performed in HeLa cells, as described in Materials and methods. HA-E6AP was expressed in E6AP KO cells, as indicated. Cells were analyzed by immunoblotting for MASTL, E6AP, and α-tubulin. ( E ) HeLa cells transfected with control or E6AP siRNA were treated with 20 μg/ml cycloheximide (CHX), as indicated. Cells were harvested and analyzed by immunoblotting for MASTL and β-actin. ( F ) HeLa cells were treated as in panel E, MASTL and β-actin protein levels were quantified, and the ratio is shown for the indicated time points after CHX treatment, after normalized to that of time 0. The mean values and standard deviations were calculated from three experiments. ( G ) WT or E6AP KO HeLa cells were transfected with HA-tagged ubiquitin for 12 hr, followed 50 μM MG132 treatment for 4 hr. Cell lysates were harvested for HA immunoprecipitation (IP) or ctr IP using blank beads. The input and IP products were analyzed by immunoblotting for MASTL and HA. ( H ) In vitro ubiquitination assay was performed using E6AP as E3 ligase, and MASTL as substrate, as described in Materials and methods. S5a was added as a control substrate. The reactions were incubated as indicated, as analyzed by immunoblotting for MASTL, ubiquitination, and S5a.

Techniques: Transfection, Control, Western Blot, Immunofluorescence, Expressing, Gene Knockout, Ubiquitin Proteomics, Immunoprecipitation, In Vitro, Incubation

( A ) HeLa cells were treated without or with E6AP siRNA. The RNA levels of E6AP and MASTL were quantified by real-time PCR. The mean values were calculated from three experiments. Statistical significance was determined using an unpaired two-tailed Student’s t test. A p-value more than 0.05 was considered non-significant (ns). ( B ) Control or E6AP knockout (KO) HeLa cells were treated without or with MG132, as indicated. The protein levels of MASTL and α-tubulin are shown by immunoblotting. ( C ) The E6AP in vitro ubiquitination assay was performed using MASTL as substrates, as in . S5a was added as a control substrate. E1/2 and E3 (E6AP) enzymes were added in the reactions, as indicated. Samples were analyzed by immunoblotting for MASTL and S5a after 90 min incubation. ( D ) In vitro ubiquitination assay was performed using E6AP as E3 ligase, and ΔN MASTL (aa 335–887) as substrate. S5a was added as a control substrate. The reactions were incubated as indicated, as analyzed by immunoblotting for MASTL and S5a.

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 were treated without or with E6AP siRNA. The RNA levels of E6AP and MASTL were quantified by real-time PCR. The mean values were calculated from three experiments. Statistical significance was determined using an unpaired two-tailed Student’s t test. A p-value more than 0.05 was considered non-significant (ns). ( B ) Control or E6AP knockout (KO) HeLa cells were treated without or with MG132, as indicated. The protein levels of MASTL and α-tubulin are shown by immunoblotting. ( C ) The E6AP in vitro ubiquitination assay was performed using MASTL as substrates, as in . S5a was added as a control substrate. E1/2 and E3 (E6AP) enzymes were added in the reactions, as indicated. Samples were analyzed by immunoblotting for MASTL and S5a after 90 min incubation. ( D ) In vitro ubiquitination assay was performed using E6AP as E3 ligase, and ΔN MASTL (aa 335–887) as substrate. S5a was added as a control substrate. The reactions were incubated as indicated, as analyzed by immunoblotting for MASTL and S5a.

Techniques: Real-time Polymerase Chain Reaction, Two Tailed Test, Control, Knock-Out, Western Blot, In Vitro, Ubiquitin Proteomics, Incubation

( 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.

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.

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

( A ) Control or MASTL knockdown HeLa cells were maintained in cell culture, and cell viability was determined by counting cell numbers. The cell numbers in days 2 and 3 were normalized to those in day 1. The mean values and standard derivations, calculated from three experiments, are shown. ( B ) Control or E6AP knockout (KO) HeLa cells were treated with 0.1 μM etoposide (ETO) for the indicated hours. Cells were analyzed by immunoblotting for phospho-ATM/ATR substrates and α-tubulin.

Journal: eLife

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

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) Control or MASTL knockdown HeLa cells were maintained in cell culture, and cell viability was determined by counting cell numbers. The cell numbers in days 2 and 3 were normalized to those in day 1. The mean values and standard derivations, calculated from three experiments, are shown. ( B ) Control or E6AP knockout (KO) HeLa cells were treated with 0.1 μM etoposide (ETO) for the indicated hours. Cells were analyzed by immunoblotting for phospho-ATM/ATR substrates and α-tubulin.

Techniques: Control, Knockdown, Cell Culture, Knock-Out, Western Blot

( 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.

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.

Techniques: Expressing, Immunoprecipitation, Control, Western Blot

( A ) The sequence alignment of the conserved E6AP Ser-218 motif in human, mouse, and Xenopus . ( B ) A phospho-specific antibody recognizing E6AP Ser-218 was generated, as described in Materials and methods. WT or E6AP knockout (KO) HEK293 cells were treated without or with 10 mM hydroxyurea (HU) for 4 hr, and analyzed by immunoblotting for phospho-E6AP Ser-218, E6AP, and α-tubulin. ( C ) HeLa cells were treated without or with 0.5 μM doxorubicin (DOX) and 5 μM KU55933 (ATMi), as indicated, for 1 hr, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-E6AP Ser-218, and α-tubulin. ( D ) HeLa cells were transfected with HA-tagged WT, S218A, or S218D E6AP. Cell lysates were harvested for immunoprecipitation (IP) assays. The input, MASTL IP, and a control IP using empty beads products were analyzed by immunoblotting for MASTL and HA. ( E ) HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in panel D. Cells were treated with or without 0.5 μM DOX for 3 hr, and harvested for IP assays. The input, HA IP, and a control IP using empty beads products were analyzed by immunoblotting for MASTL, phospho-E6AP Ser-218, and HA. ( F ) E6AP KO HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in panel D. Cells were treated with or without 0.5 μM DOX, incubated as indicated, and harvested for immunoblotting for MASTL and α-tubulin.

Journal: eLife

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

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) The sequence alignment of the conserved E6AP Ser-218 motif in human, mouse, and Xenopus . ( B ) A phospho-specific antibody recognizing E6AP Ser-218 was generated, as described in Materials and methods. WT or E6AP knockout (KO) HEK293 cells were treated without or with 10 mM hydroxyurea (HU) for 4 hr, and analyzed by immunoblotting for phospho-E6AP Ser-218, E6AP, and α-tubulin. ( C ) HeLa cells were treated without or with 0.5 μM doxorubicin (DOX) and 5 μM KU55933 (ATMi), as indicated, for 1 hr, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-E6AP Ser-218, and α-tubulin. ( D ) HeLa cells were transfected with HA-tagged WT, S218A, or S218D E6AP. Cell lysates were harvested for immunoprecipitation (IP) assays. The input, MASTL IP, and a control IP using empty beads products were analyzed by immunoblotting for MASTL and HA. ( E ) HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in panel D. Cells were treated with or without 0.5 μM DOX for 3 hr, and harvested for IP assays. The input, HA IP, and a control IP using empty beads products were analyzed by immunoblotting for MASTL, phospho-E6AP Ser-218, and HA. ( F ) E6AP KO HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in panel D. Cells were treated with or without 0.5 μM DOX, incubated as indicated, and harvested for immunoblotting for MASTL and α-tubulin.

Techniques: Sequencing, Generated, Knock-Out, Western Blot, Transfection, Immunoprecipitation, Control, Incubation

( A ) WT or E6AP knockout HEK293 cells were treated without or with doxorubicin (DOX, 0.5 μM) for 4 hr. Cells were harvested and analyzed by immunoblotting for phospho-E6AP Ser-218 and α-tubulin. ( B ) HEK293 cells were treated without or with DOX (0.5 μM), or ATM inhibitor (KU55933, 10 μM), for 4 hr, and analyzed by immunoblotting. ( C ) HeLa cells were treated with hydroxyurea (HU, 10 mM) combined with ATM/ATR inhibitor (caffeine, 4 mM) or ATM inhibitor (KU55933, 10 μM), for 12 hr, as indicated. Cells were harvested and analyzed by immunoblotting.

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 HEK293 cells were treated without or with doxorubicin (DOX, 0.5 μM) for 4 hr. Cells were harvested and analyzed by immunoblotting for phospho-E6AP Ser-218 and α-tubulin. ( B ) HEK293 cells were treated without or with DOX (0.5 μM), or ATM inhibitor (KU55933, 10 μM), for 4 hr, and analyzed by immunoblotting. ( C ) HeLa cells were treated with hydroxyurea (HU, 10 mM) combined with ATM/ATR inhibitor (caffeine, 4 mM) or ATM inhibitor (KU55933, 10 μM), for 12 hr, as indicated. Cells were harvested and analyzed by immunoblotting.

Techniques: Knock-Out, Western Blot

( A ) E6AP knockout (KO) HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in . Cells were treated with 0.1 μM etoposide (ETO) for 18 hr, and released in fresh medium for recovery. Cells were then 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<0.01, n>500 cell numbers/measurement). ( B ) E6AP KO HeLa cells expressing HA-tagged WT or S218A E6AP, 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. Cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS). ( C ) WT or S218A E6AP was expressed in E6AP KO HEK293 cells. Cells were treated without or with 0.1 μM ETO for 18 hr, released in fresh medium for recovery, and incubated as indicated. Cells were analyzed by immunoblotting for phospho-cyclin-dependent kinase (CDK) substrates and histone H3. ( D ) WT or S218A E6AP was expressed in E6AP KO HEK293 cells, as in panel C. Cells were treated without or with 1 μM CPT for 90 min, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, and α-tubulin.

Journal: eLife

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

doi: 10.7554/eLife.86976

Figure Lengend Snippet: ( A ) E6AP knockout (KO) HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in . Cells were treated with 0.1 μM etoposide (ETO) for 18 hr, and released in fresh medium for recovery. Cells were then 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<0.01, n>500 cell numbers/measurement). ( B ) E6AP KO HeLa cells expressing HA-tagged WT or S218A E6AP, 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. Cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS). ( C ) WT or S218A E6AP was expressed in E6AP KO HEK293 cells. Cells were treated without or with 0.1 μM ETO for 18 hr, released in fresh medium for recovery, and incubated as indicated. Cells were analyzed by immunoblotting for phospho-cyclin-dependent kinase (CDK) substrates and histone H3. ( D ) WT or S218A E6AP was expressed in E6AP KO HEK293 cells, as in panel C. Cells were treated without or with 1 μM CPT for 90 min, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, and α-tubulin.

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

DNA damage induces ATM/ATR activation and checkpoint signaling. At this stage (the initial DNA damage sensing/signaling), MASTL expression is normal (not yet upregulated). Activated ATM/ATR also phosphorylates E6AP Ser-218, leading to the dissociation of E6AP from MASTL and reduced MASTL degradation. The subsequent accumulation of MASTL, at upregulated levels several hours after DNA damage, promotes de-activation of the DNA damage checkpoint and initiates cell cycle resumption by inhibiting dephosphorylation of cyclin-dependent kinase (CDK) substrates.

Journal: eLife

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

doi: 10.7554/eLife.86976

Figure Lengend Snippet: DNA damage induces ATM/ATR activation and checkpoint signaling. At this stage (the initial DNA damage sensing/signaling), MASTL expression is normal (not yet upregulated). Activated ATM/ATR also phosphorylates E6AP Ser-218, leading to the dissociation of E6AP from MASTL and reduced MASTL degradation. The subsequent accumulation of MASTL, at upregulated levels several hours after DNA damage, promotes de-activation of the DNA damage checkpoint and initiates cell cycle resumption by inhibiting dephosphorylation of cyclin-dependent kinase (CDK) substrates.

Techniques: Activation Assay, Expressing, De-Phosphorylation Assay

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