Figure 5: SENP1 serves as a desumoylating enzyme for DBC1.

(a) HisMax-DBC1 was expressed in HEK293T cells 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=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=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=3).