anti rad18 d2b8 xp  (Cell Signaling Technology Inc)

Journal: Protein & Cell

Article Title: NEDDylation antagonizes ubiquitination of proliferating cell nuclear antigen and regulates the recruitment of polymerase η in response to oxidative DNA damage

doi: 10.1007/s13238-017-0455-x

Figure Lengend Snippet: RAD18 acts as an E3 ligase of PCNA NEDDylation . (A) Analysis of the interaction between RAD18 and UBC12. HEK293T cells were lysed in NP-40 lysis buffer, and sonicated for 15 times, then immunoprecipitation was performed using antibody against RAD18. (B and C) Immunoblotting analysis of the effect of RAD18 on PCNA NEDDylation in His-tagged NEDD8 conjugates from HEK293T WT (B) or Ubc12 −/− cells (C). HEK293T WT cells or Ubc12 −/− cells were transfected with the corresponding plasmids, and PCNA NEDDylation or ubiquitination was analyzed by Ni-PD assay and Western blotting with indicated antibodies. (D) Analysis of PCNA NEDDylation by in vitro NEDDylation assay. GST-PCNA and His-SUMO-RAD18 were incubated with E1, UBC12 or RAD6B, and NEDD8 at 37°C for 1 h, and then the reactions were terminated by adding SDS buffer and detected by Western blotting with indicated antibody. (E) Immunoblotting analysis of PCNA NEDDylation in Rad18 −/− cells. HEK293T WT or Rad18 −/− cells were transfected with indicated plasmids, and PCNA NEDDylation was detected as described above. Fold change of PCNA modification intensity was analyzed using odyssey imaging systems (LI-COR, Lincoln, Nebraska USA)

Article Snippet: PCNA mouse monoclonal antibody (BE0029) and histone H3 monoclonal antibody (BE3015) were purchased from EASYBIO (Seoul, Korea); RAD18 rabbit monoclonal antibody (D2B8) was purchased from CST (Danvers, MA, USA); polη rabbit monoclonal antibody (BS6695) was purchased from Bioworld (St. Louis Park, MN, USA); NEDD8 rabbit monoclonal antibody (Y297) was purchased from Epitomics (Cambridge, MA, USA); HA mouse monoclonal antibody (H9658) and Flag mouse monoclonal antibody (F3165) were purchased from Sigma-Aldrich (St. Louis, MO, USA); His mouse monoclonal antibody (D291-3) and actin rabbit monoclonal antibody (PM053) were purchased from MBL (Nagano, Japan); and NEDP1 monoclonal antibody (F1512) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Lysis, Sonication, Immunoprecipitation, Western Blot, Transfection, Ubiquitin Proteomics, In Vitro, Incubation, Modification, Imaging

Journal: Protein & Cell

Article Title: NEDDylation antagonizes ubiquitination of proliferating cell nuclear antigen and regulates the recruitment of polymerase η in response to oxidative DNA damage

doi: 10.1007/s13238-017-0455-x

Figure Lengend Snippet: RAD18 is essential for the accumulation of NEDDylated PCNA under oxidative stress . (A) Immunoblotting analysis of the effect of RAD18 on oxidative stress-induced PCNA ubiquitination and NEDDylation in the TIF from WT or Rad18 −/− HEK293T cells. Cells were treated with 800 μmol/L H 2 O 2 for the indicated time, and then the TIF was isolated and subjected to staining with anti-PCNA antibody. (B and C) Immunoblotting analysis of the effect of RAD18 on PCNA NEDDylation under H 2 O 2 -induced oxidative stress. WT and Rad18 −/− HEK293T cells were transfected with the indicated plasmids, and at 36 h after transfection, the cells were treated with or without 800 μmol/L H 2 O 2 for 30 min before PCNA NEDDylation was detected as described above. (D) Immunofluorescence analysis of RAD18 localization after H 2 O 2 treatment. HeLa cells were treated with or without 800 μmol/L H 2 O 2 for 30 min, and then endogenous RAD18 was examined by immunofluorescence staining using anti-RAD18 antibody. The scale bar is 10 μm. (E) Analysis of the effect of RAD18 on cell survival against oxidative stress-induced DNA damage. HeLa WT and Rad18 −/− cells were cultured and treated with the indicated doses of H 2 O 2 , and then the cells were cultured for 12 days before colony counting. The error bars represented the standard error of the mean (SEM). Anti-RAD18 antibody was used for Western blotting analysis

Article Snippet: PCNA mouse monoclonal antibody (BE0029) and histone H3 monoclonal antibody (BE3015) were purchased from EASYBIO (Seoul, Korea); RAD18 rabbit monoclonal antibody (D2B8) was purchased from CST (Danvers, MA, USA); polη rabbit monoclonal antibody (BS6695) was purchased from Bioworld (St. Louis Park, MN, USA); NEDD8 rabbit monoclonal antibody (Y297) was purchased from Epitomics (Cambridge, MA, USA); HA mouse monoclonal antibody (H9658) and Flag mouse monoclonal antibody (F3165) were purchased from Sigma-Aldrich (St. Louis, MO, USA); His mouse monoclonal antibody (D291-3) and actin rabbit monoclonal antibody (PM053) were purchased from MBL (Nagano, Japan); and NEDP1 monoclonal antibody (F1512) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Western Blot, Ubiquitin Proteomics, Isolation, Staining, Transfection, Immunofluorescence, Cell Culture

Journal: Protein & Cell

Article Title: NEDDylation antagonizes ubiquitination of proliferating cell nuclear antigen and regulates the recruitment of polymerase η in response to oxidative DNA damage

doi: 10.1007/s13238-017-0455-x

Figure Lengend Snippet: Model of PCNA NEDDylation . (A) Illustration of PCNA NEDDylation cascade. In an ATP-dependent manner, a NEDD8-adenylate intermediate is produced to form a NEDD8-E1 (APPBP1-UBA3) complex. Then NEDD8 is transferred from the E1 to the E2 (UBC12), which combines with the E3 ligase RAD18. NEDD8 is subsequently conjugated to the Lys164 of PCNA. In this process, MLN4924 can inhibit the function of E1, thus impeding PCNA NEDDylation, and NEDP1 acts as the deNEDDylase of PCNA. (B) The molecular function of PCNA NEDDylation in regulating its monoubiquitination and TLS. In cells without stress, PCNA acts as a clamp to recruiting polε for the normal replication process. When cells were under oxidative DNA damage, monoubiquitinated PCNA recruits TLS polymerase polη to bypass the lesions. Meanwhile, NEDP1 disassociates from PCNA and PCNA is NEDDylated. PCNA NEDDylation antagonizes its monoubiquitination and leads to defective PCNA-polη interaction and reduced TLS activity

Article Snippet: PCNA mouse monoclonal antibody (BE0029) and histone H3 monoclonal antibody (BE3015) were purchased from EASYBIO (Seoul, Korea); RAD18 rabbit monoclonal antibody (D2B8) was purchased from CST (Danvers, MA, USA); polη rabbit monoclonal antibody (BS6695) was purchased from Bioworld (St. Louis Park, MN, USA); NEDD8 rabbit monoclonal antibody (Y297) was purchased from Epitomics (Cambridge, MA, USA); HA mouse monoclonal antibody (H9658) and Flag mouse monoclonal antibody (F3165) were purchased from Sigma-Aldrich (St. Louis, MO, USA); His mouse monoclonal antibody (D291-3) and actin rabbit monoclonal antibody (PM053) were purchased from MBL (Nagano, Japan); and NEDP1 monoclonal antibody (F1512) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Produced, Activity Assay