FANCD2 noncovalently interacts with ubiquitin. (A) WCLs from FA-D2 cells expressing WT or mutant FANCD2 were incubated with ubiquitin or protein G-conjugated sepharose and bound proteins resolved and immunoblotted with antibodies against FANCD2. * indicates nonspecific band; WCL, whole-cell lysate; Ub-seph., ubiquitin-conjugated sepharose; and G-seph., protein G-conjugated sepharose. (B) WCLs from HeLa cells expressing WT or mutant FANCD2-V5 were incubated with ubiquitin or SIM-conjugated sepharose and bound proteins resolved and immunoblotted with antibodies against V5. (C) Bacterially purified fragments of FANCD2 were incubated with ubiquitin-conjugated sepharose and bound proteins resolved and immunoblotted with antibodies against V5. (D) Bacterial WCLs expressing GST-Empty or GST-tagged WT FANCD2 solenoid 1 (D2-S1 WT), FANCD2 S1 P204A (D2-S1 P-A), or FANCD2 S1 LL234AA (D2-S1 LL-AA) were incubated with ubiquitin-conjugated sepharose and bound proteins resolved and immunoblotted with antibodies against FANCD2.