FIGURE 4.
MNNG-induced p21 degradation requires Cdt2 and PCNA. A, RNAi-mediated knockdown of the ubiquitin ligase adaptor subunit Cdt2 blocked p21 degradation in MNNG-treated (M) and UV-irradiated HeLa cells. B, a p21 mutant unable to bind PCNA (p21PCNA−) was not degraded after MNNG treatment. HeLa cells were transfected with plasmids expressing the WT or mutant p21 proteins as indicated. The recombinant p21 proteins have an N-terminal HA epitope tag resulting in a higher molecular weight than wild-type p21. C, MSH6 and Cdt2 co-immunoprecipitated with PCNA (PCNA IP) after MNNG treatment when protein complexes had been stabilized by chemical cross-linking with DSP. D, reverse immunoprecipitation with Cdt2 antibody (Cdt2 IP) demonstrated the same complex.