Figure 3.
PIDD regulates the PCNA-p21 interaction in response to UV-C irradiation. (a–e) Where indicated, endogenous p21 interaction with PCNA was analysed by anti-VSV IP, endogenous PCNA interaction with Flag-tagged PIDD was analysed after anti-Flag IP, and p21 degradation and PIDD expression were checked in total extracts. (a) PIDD upregulation correlates with PCNA-p21 detachment in the course of UV-C: 293T cells were transfected with VSV-tagged PCNA and subjected to different times of 50 J/m2 UV-C irradiation. (b) PIDD overexpression is able to induce p21 detachment from PCNA: 293T cells were cotransfected with VSV-tagged PCNA and increasing amounts of Flag-tagged PIDD (the number indicates the ratio of Flag-PIDD plasmid transfected compared with the VSV-PCNA plasmid). (c) PCNA trimerization is not required for interaction with PIDD: 293T cells were cotransfected with differents constructs of VSV-tagged PCNA and Flag-tagged PIDD where indicated. See also Supplementary Figures S3a–c. (d) PIDD overexpression reduces p21-PCNA interaction in response to UV-C irradiation: 293T cells were transfected with VSV-tagged PCNA and with Flag-tagged PIDD and subjected to 100 J/m2 UV-C irradiation for 1 h where indicated. (e) PIDD deficiency leads to sustained p21-PCNA interaction in response to UV-C irradiation: 293T cells stably knocked-down for PIDD or the corresponding control cells, were transfected with VSV-tagged PCNA and subjected to 50 J/m2 UV-C irradiation for 30 min where indicated. All the figures are representative of at least three repetitions