Figure 4: DNA damage induces p53 ISGylation.
From: Positive feedback regulation of p53 transactivity by DNA damage-induced ISG15 modification
(a) HA-p53 and Flag-ISG15, Myc-UBE1L (E1) and Myc-UBCH8 (E2) were expressed in HEK293T cells with or without Flag-UBP43. The cell lysates were subjected to immunoprecipitation (IP) with anti-HA antibody followed by immunoblot with anti-Flag or anti-HA antibody. They (lysates) were also directly probed with anti-HA, anti-Flag or anti-Myc antibody. (b) p53+/+ HCT116 cells that had been transfected with shNS or shISG15 were treated with doxorubicin (DOX) or camptothecin (CPT) for 24 h. They were also irradiated 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. They were also directly probed with respective antibodies. (c) Deletions of p53 (pΔ1–pΔ4) were tagged with HisMax to their N-termini, and expressed in HEK293T cells with Flag-ISG15, Myc-UBE1L and Myc-UBCH8. The cell lysates were subjected to pull-down with NTA resins followed by immunoblot with anti-Flag antibody. (d) Wild-type p53 (Wt) or its K-to-R mutants were expressed in HEK293T cells with Flag-ISG15, Myc-UBE1L and Myc-UBCH8. The cell lysates were subjected to immunoprecipitation with anti-Xpress antibody followed by immunoblot with anti-Flag or anti-Xpress antibody. (e) HCT116 (p53+/+) cells were transfected with shNS or shISG15. After exposure to ultraviolet, the cells were subjected to incubation with 0.2 mg ml−1 cycloheximide (CHX) for increasing periods followed by immunoblot analysis. (f) Experiments in e were repeated and the band intensities were scanned by using a densitometer and normalized by those of GAPDH. The normalized densities seen at ‘0’ time points were expressed as 1.0 and the others were expressed as its relative values. Error bar, ±s.d. (n=3).
