SDE2-UBL undergoes CRL4^CDT2-dependent proteolysis.

<p><b>(A)</b> HeLa cells expressing full-length GFP-SDE2 were left untreated or treated with 40 J/m² ultraviolet C (UVC), 2 mM hydroxyurea (HU), or 1 μM mitomycin C (MMC) for the indicated times, and N-terminal GFP-UBL levels were analyzed by Western blotting. <b>(B)</b> HeLa cells exponentially growing or synchronized by nocodazole were collected at the indicated times following mitotic shake-off and analyzed by Western blotting. Cell cycle progression was analyzed by flow cytometry in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006465#pgen.1006465.s004" target="_blank">S3C Fig</a>. <b>(C)</b> (left) Full-length GFP-SDE2 expressing HeLa cells transfected with siRNA CDT2 (vs. control) were treated with 2 mM HU for the indicated times and analyzed by Western blotting. (right) Quantification of immunoblots by Image J. <b>(D)</b> HeLa cells expressing full-length GFP-SDE2 were transfected with siRNA control or CDT2 for 48 h and treated with 50 μg/mL cycloheximide (CHX) for the indicated times. The level of GFP-UBL was analyzed by Western blotting. <b>(E)</b> HeLa cells treated with DMSO or 1 μM MLN4924 were treated with 50 μg/mL CHX for the indicated times, and GFP-UBL levels were analyzed by Western blotting. <b>(F)</b> HeLa cells expressing GFP-SDE2 were transfected with HA-CDT2 plasmid (vs. empty vector), and GFP-UBL levels were analyzed by Western blotting. <b>(G)</b> 293T cells transfected with the indicated plasmids were treated with 10 μM MG132 for 4 h, lysed in a denaturing condition, and incubated with Ni-NTA agarose for the <i>in vivo</i> ubiquitination assay. <b>(H)</b> 293T cells transfected with indicated siRNAs and plasmids were subjected to the <i>in vivo</i> ubiquitination assay.