CRL4A/4B^CDT2 modulates RAD18 binding to chromatin and PCNA monoubiquitylation.

<p>(A) HeLa cell total protein lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies; (AS) exponentially growing HeLa cells transfected with the indicate siRNAs, mock or UV-irradiated; (S) HeLa cells were synchronized in mid-S-phase after transfection with the indicated siRNAs as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060000#pone-0060000-g003" target="_blank">Figure 3A</a>. HMW and LMW indicates respectively slow and high migrating CDT2; Ub1 indicates mono-ubiquitin-PCNA; ° indicates a not specific band. (B) HeLa cells were synchronized in mid-S-phase, protein-protein cross-linked and harvested. Total cell protein extract was prepared. Cell lysate was subjected to immunoprecipitation with serum as control or with the indicated antibodies. Total cells lysate (input,10%) and immunoprecipitates were immunoblotted with the indicated antibodies (C) (D) HeLa cells were transfected with the indicated siRNAs as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060000#pone-0060000-g003" target="_blank">Figure 3A</a>, synchronized and harvested in mid-S-phase. Total protein extract were fractionated into soluble and chromatin-bound fractions, resolved by SDS-PAGE and immunoblotted with the indicated antibodies (D) MG132 was added for two hours prior to harvesting where indicated.