FAAP20 binds ubiquitin in vitro. (A) Immunoblot showing mono-ubiquitin (Ub), K-48 and K-63–linked ubiquitin chains. Interaction of M2-agarose bound _His6-FLAGFAAP20 was tested with mono-ubiquitin, K-48 and K-63–linked ubiquitin chains. FAAP20 binds K-63 linked ubiquitin chains (lane 8), but not mono-ubiquitin (lane 6) or K-48 linked (lane 7). Mutation in the UBZ domain abolished binding (lane 9). RAD18 served as a positive control (lanes 3-5). (B) Immunoblot showing K-63- linked ubiquitin chains. Interaction of M2-agarose bound _His6-FLAGFAAP20 WT (Wt) and various point mutants was tested with K-63–linked ubiquitin chains. FAAP20 constructs with single residue mutations in the UBZ domain, D164A mutation (lane 3) abolishes binding of FAAP20, whereas D162A (lane 2) and E173A (lane 4) bind K-63–linked chains. (C) Immunoblot showing no apparent difference between WT FAAP20 and UBZ mutant (C170A or D164A) binding to core-complex proteins. (D) Interaction of M2-agarose bound _His6-FLAGFAAP20 alone (−) or in complex with _MBPFANCA_1065-1455 (+) was tested with mono-ubiquitin, K-48 and K-63–linked ubiquitin chains. No apparent difference was found in binding of FAAP20 with K-63–linked chains in the absence or presence of FANCA.