Journal: Nature communications

Article Title: Modification of DBC1 by SUMO2/3 is crucial for p53-mediated apoptosis in response to DNA damage.

doi: 10.1038/ncomms6483

Figure Lengend Snippet: Figure 5 | SENP1 serves as a desumoylating enzyme for DBC1. (a) HisMax-DBC1 was expressed in HEK293Tcells with each of SENP1, 2, 5 and 7 (left) or SENP3 and 6 (right). Cell lysates were subjected to immunoprecipitation with anti-Myc (left) and anti-Flag antibodies (right) followed by immunoblot analysis. (b) HeLa cells incubated with and without etoposide for 36 h were subjected to immunoprecipitation with anti-DBC1 antibody followed by immunoblot analysis (left). The band intensities of SENP1 from immunoprecipitation were quantified, and that seen without etoposide was expressed as 1.0 and the others were expressed as its relative values (right). Error bar, ±s.d. (n ¼ 3). (c) HisMax-DBC1, Flag-Ubc9 and Flag-SUMO3 were expressed in HEK293T cells with Myc-tagged SENP1 or its C603S mutant. Cell lysates were subjected to pull-down with NTA-resins followed by immunoblot analysis. (d) HeLa cells expressing nonspecific shRNA (shNS) or shSENP1 were incubated with and without etoposide for 36 h. Their lysates were then treated as in b (left). The band intensities of DBC1-SUMO2/3 (top) and SIRT1 from immunoprecipitation and acetylated p53 from lysates were quantified. The intensities of DBC1-SUMO2/3 and acetylated p53 seen with both shNS and etoposide and that of SIRT1 seen with shNS alone were expressed as 1.0 and the others were expressed as its relative values (right). Error bar, ±s.d. (n ¼ 3). (e) shNS and shSENP1 were expressed in HeLa cells with and without Myc-SENP1. They were incubated with and without etoposide for 36 h. Cell lysates were subjected to immunoprecipitation with anti-SIRT1 antibody followed by immunoblot analysis. ‘i’ and ‘e’ denote shRNA insensitive and endogenous, respectively (left). The band intensities of DBC1 from immunoprecipitation and acetylated p53 from lysates were quantified. The intensity of DBC1 seen with shNS only and that of acetylaed-p53 seen with shNS and etoposide were expressed as 1.0 and the others were expressed as its relative values (right). Error bar, ±s.d. (n ¼ 3).

Article Snippet: Antibodies against p53 (catalogue#: DO-1), GAPDH (2D4A7), Ubc9 (N-15), DBC1 (H-2) and SENP1 (C-12) were purchased from Santa Cruz.

Techniques: Immunoprecipitation, Western Blot, Incubation, Mutagenesis, Expressing, shRNA

Journal: Nature communications

Article Title: Modification of DBC1 by SUMO2/3 is crucial for p53-mediated apoptosis in response to DNA damage.

doi: 10.1038/ncomms6483

Figure Lengend Snippet: Figure 6 | DBC1 phosphorylation promotes its sumoylation. (a) Flag-tagged SUMO3 and Ubc9 were expressed in HEK293T cells with HisMax-tagged DBC1, T454A or T454D. Cell lysates were subjected to pull-down with NTA-resins followed by immunoblot analysis (left). The band intensities of DBC1-SUMO3 (top) and SIRT1 from immunoprecipitation were quantified, and those seen with HisMax-DBC1, Myc-SIRT1 and Flag-tagged SUMO3 and Ubc9 were expressed as 1.0 and the others were expressed as its relative values (right). Error bar, ±s.d. (n ¼ 3). (b) shRNA-insensitive Flag-tagged DBC1, T454A or T454D was expressed in HeLa cells that had been stably transfected with nonspecific shRNA (shNS) or shDBC1. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody followed by immunoblot analysis (left). The band intensities of PIAS3 and SENP1 from immunoprecipitation were quantified, and those seen with both shDBC1 and Flag-DBC1 were expressed as 1.0 and the others were expressed as its relative values (right). Error bar, ±s.d. (n ¼ 3). (c) HeLa cells were incubated with etoposide and/or 5 mM caffeine for 36 h. Cell lysates were subjected to immunoprecipitation with anti-DBC1 antibody followed by immunoblot analysis.

Article Snippet: Antibodies against p53 (catalogue#: DO-1), GAPDH (2D4A7), Ubc9 (N-15), DBC1 (H-2) and SENP1 (C-12) were purchased from Santa Cruz.

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, shRNA, Stable Transfection, Transfection, Incubation

Journal: Molecular and Cellular Biology

Article Title: Sumoylation of the Novel Protein hRIPβ Is Involved in Replication Protein A Deposition in PML Nuclear Bodies

doi: 10.1128/mcb.25.18.8202-8214.2005

Figure Lengend Snippet: FIG. 7. Sumoylation of hRIP is regulated by UV irradiation and the normal cell cycle. (A) Panel i, UV irradiation desumoylates hRIP. HA-hRIP was transiently expressed in HEK293T cells. After 24 h, the cells were either left untreated (lane 1) or irradiated with 50 J/m2 of UV (lanes 2 and 3). The irradiated cells were harvested at the indicated times and the cell lysates blotted with anti-HA antibody. Panel ii, the sumoylation of endogenous (endo.) hRIP upon UV irradiation was measured to confirm the results. (B) SENP1 desumoylates hRIP. Panel i, cells were transfected with a plasmid encoding HA-hRIP in the presence or absence of a plasmid encoding His-SENP1. After 48 h, the cells were collected and the cell lysates blotted with anti-HA antibody (upper panel) or anti-His antibody (lower panel). Panel ii, the transiently expressed SENP1 desumoylated the endogenous hRIP. Panel iii, SENP1 siRNA was used to inhibit SENP1-mediated desumoylation of hRIP. Panel iv, SENP1 siRNA treatment decreased the endogenous SENP1 protein. (C) UV irradiation disrupts the interaction between hRIP and RPA. hRIP and RPA were expressed in HEK293T cells grown on glass coverslips. After 24 h, the cells were either left untreated or irradiated with 50 J/m2

Article Snippet: Antibodies for RPA p70 and p32 were generously provided by B. Stillman (8), while antibodies for PML and lamin were purchased from Santa Cruz (Santa Cruz, CA) and antibody for SENP1 was purchased from IMGENEX (San Diego, CA).

Techniques: Irradiation, Transfection, Plasmid Preparation