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. 2013 Sep 10;288(42):30509–30514. doi: 10.1074/jbc.C113.505586

FIGURE 1.

FIGURE 1.

CRL4Cdt2 depletion abrogates the degradation of p12 by UV irradiation. A and B, HeLa cells were transfected with the indicated siRNA and analyzed by immunoblotting with anti-p12, anti-Cdt2, anti-p125, anti-DDB1, anti-Cul4A/B, or anti-PCNA antibody. C, HeLa cells were transfected with si-Cdt2, si-Cul4A/B, or si-DDB1. After 48 h, cell cycle was analyzed by propidium iodide staining followed by flow cytometry. D, HeLa cells stably expressing HA-tagged Cdt2 or Cdt2-R246A were transfected with si-Cdt2 targeting the ORF (ORF) or si-Cdt2 targeting the 3′-UTR (3′-UTR). Cells were irradiated with UV and lysed 1 h after irradiation. Protein lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-PCNA, anti-Cdt2, or anti-p12 antibody. Exo., exogenous; Endo., endogenous. E, immunopurified CRL4Cdt2 complexes (E3) were mixed with p12WT, E1, and UbcH5 (E2). Ubiquitinated p12 (Ub-p12) was detected by Western blotting after separating the reaction products by SDS-PAGE.