FAAP20 interact with FANCA in vivo and in vitro. (A) Schematic of FANCA deletion constructs expressed in HSC72 cells. Nuclear localization signal (NLS), leucine-zipper (LZ) motif and C-terminal FLAG tag are shown. (B) Immunoblot of FLAG IP showing that full-length FANCA and the N-terminal deletion interact with FAAP20, but the C-terminal deletion shows weak or no interaction. (C) Immunoblot showing FANCD2 mono-ubiquitination in MMC treated (MMC) or untreated (UT) HSC72 cells expressing various FANCA deletion fragments. Although full-length FANCA corrected the FANCD2 monoubiquitination defect in HSC72 cells, all 3 deletion constructs failed to do so. (D) Schematic of FANCA deletion constructs expressed in E coli cells. MBP is fused to N-terminus. (E) Schematic of FAAP20 full-length construct expressed in E coli. (F) Top panel: Coomassie-stained gel showing FAAP20 and FANCA purified from E coli cell lysate expressing _His6-FLAGFAAP20 or _MBPFANCA fragment or both using M2 agarose (FAAP20 target) or amylose (FANCA target) beads. _MBPFANCA_1065-1101 construct with the leucine-zipper motif failed to interact with FAAP20. _MBPFANCA_1095-1455 and _MBPFANCA_1095-1200 lacking the motif were able to interact with FAAP20. Bottom panel: immunoblot analysis using anti-MBP or anti-FAAP20 confirmed the identity of the bands as FANCA or FAAP20, respectively.