Fig. 4

T457A impairs CRL4^CDT2-mediated Polη polyubiqitination. a 293T cells were transfected with siDDB1 or siNC oligos. Seventy-two hours later, the cells were harvested and the protein levels of Polη and DDB1 were detected by western blotting. Tubulin: loading control. b Flag empty vector or Flag-Polη and HA-Ub were transfected in siNC- or siDDB1-treated 293T cells. The cells lysates were immunoprecipitated by anti-Flag M2 beads and analyzed by immunoblotting with anti-Ubiquitin and anti-Flag antibodies. SE short exposure, LE long exposure. c, d WT or T457A GFP-Polη and HA-Ub were transfected into siDDB1-treated c or siCDT2-treated d 293T cells. The lysates were immunoprecipitated using GFP-Trap A. The immunoprecipitates were analyzed via western blotting using anti-GFP and anti-Ubiquitin antibodies. The protein levels of DDB1 and CDT2 were detected by immunoblotting. WCE whole cell lysate. Tubulin: loading control. Asterisk denote non-specific signal. e WT or T457A GFP-Polη and HA-Ub were transfected into siDDB1-treated 293T cells. The chromatin fractions (CF) were harvested followed by immunoprecipitation with GFP-Trap A. The immunoprecipitates were analyzed as in c. f WT or T457A GFP-Polη and HA-Ub were transfected into p97 siRNA (sip97)-treated 293T cells. The chromatin fractions were immunoprecipitated and analyzed as in c. The p97 protein level was detected by western blot. g Flag-DDB1 and GFP-Polη (WT, T457A, or 5A) were transfected into 293T cells. The lysates were immunoprecipitated using GFP-Trap A. The immunoprecipitates and inputs were examined via western blotting using anti-GFP and anti-Flag antibodies. h Flag empty vector or Flag-CDT2 and GFP-Polη (WT or T457A) were transfected into 293T cells. The lysates were immunoprecipitated and analyzed as in g