Figure 4. p21 degradation during MVM requires interaction with PCNA and the CRL4^Cdt2 ligase complex.

A) Illustration of the p21 PIP/degron region (amino acid 136 to 151) of wild-type murine p21 (p21^WT), p21^Δdegron, and p21^ΔPIP. Mutations are shown in red. B) p21^Δdegron does not interact with the CRL4^Cdt2 complex. 293T were cells transfected with constructs expressing FLAG-tagged p21^WT (lane 2), p21^Δdegron (lane 3) or control plasmid (lane 1). Cells were harvested at 48 hr. Lysates were immunoprecipitated using with FLAG antibody and blotted against the indicated proteins. C) MVM degradation of p21 requires its interaction with the CRL4^Cdt2 ligase complex. Murine A9 cell lines stably expressing p21^WT and p21^Δdegron were generated as described. Cells were parasynchronized, released into complete media and mock-infected or infected with MVM at an MOI of 10. At 20 hr pi cells were treated with doxycycline to induce p21 expression. Cells were harvested 6 hrs later and processed for western blotting using the indicated antibodies. D) p21^ΔPCNA does not interact with PCNA. Experiment performed as for Figure 4B. E) MVM degradation of p21 requires its interaction with PCNA. Experiment performed as for 4C.