Mutation of the CUE domain impairs FANCD2 protein stability. (A) Using site-directed mutagenesis, 3 CUE domain missense mutations were generated. (B) WCLs from COS-7 cells expressing WT or mutant FANCD2 were resolved and immunoblotted with antibodies against FANCD2, V5, and α-tubulin (TUB). (C) WCLs from FA-D2 cells reconstituted with WT or mutant FANCD2 were resolved and immunoblotted with antibodies against FANCD2 and tubulin. h indicates hours. (D) COS-7 cells were transfected with WT or mutant FANCD2. Forty-eight hours later cells were untreated or treated with 10μM MG132 or 35μM cyclohexamide (CHX). Pellets were collected at the indicated times and WCLs generated. Proteins were resolved and immunoblotted with antibodies against V5 and α-tubulin.