Figure 6: DBC1 phosphorylation promotes its sumoylation.
From: Modification of DBC1 by SUMO2/3 is crucial for p53-mediated apoptosis in response to DNA damage
(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=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=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.
