Figure 5: EFP serves as a p53-specific ISG15 E3 ligase.
From: Positive feedback regulation of p53 transactivity by DNA damage-induced ISG15 modification
(a) HA-EFP or its inactive C13/16S mutant was expressed in HEK293T cells with HisMax-p53. The cell lysates were subjected to immunoprecipitation with anti-HA antibody or pull-down with NTA resins followed by immunoblot with anti-Xpress or anti-HA antibody. The asterisk indicates IgG heavy chain. (b) HisMax-p53, Flag-ISG15, Myc-UBE1L (E1) and Myc-UBCH8 (E2) were expressed in HEK293T cells with Myc-tagged EFP or its C13/16S mutant. The cell lysates were subjected to immunoprecipitation with anti-Xpress antibody followed by immunoblot anti-Flag or anti-Xpress antibody. (c) p53+/+ HCT116 cells that had been transfected with shNS or shEFP were treated with doxorubicin or camptothecin for 24 h. They were also treated with ultraviolet (UV), and then incubated for 24 h. The cell lysates were subjected to immunoprecipitation with anti-p53 antibody followed by immunoblot with anti-ISG15 antibody. (d) Myc-HERC5 was expressed in HEK293T cells with HisMax-p53. The cell lysates were subjected to pull-down with NTA resins followed by immunoblot with anti-Myc or anti-Xpress antibody. (e) After treatment with DNA-damaging agents, the HCT116 cells were subjected to incubation for 24 h followed by immunoblot with anti-HERC5, anti-EFP or anti-p53 antibody. (f) p53+/+ HCT116 cells expressing shNS or shHERC5 were exposed to ultraviolet. After incubation for 24 h, the cells were subjected to immunoprecipitation with anti-p53 followed by immunoblot with anti-ISG15 antibody.
