FANCA is required for stability of FAAP20. (A) Immunoblot showing levels of FANCA, FAAP20, and FANCG in various FA-patient-derived cells defective in one of the complementation groups (indicated above each lane); actin serves as loading control. In the absence of FANCA, FAAP20 amounts were negligible. (B) Immunoblot showing levels of FANCA, FAAP20, and FANCG from HSC93 WT cells and various FA-A patient-derived cells obtained from IFAR. FAAP20 amounts were negligible in the absence of FANCA. (C) Immunoblot showing that FAAP20 and FANCG but not FANCM are stabilized in FANCA-overexpressing HSC72 cells. A nonspecific band is indicated by an asterisk. (D) Immunoblot showing levels of FANCA and FAAP20 in IPs of HSC72 and HSC72:FANCA_FLAG cell extracts. Negligible amount of FAAP20 was immunoprecipitated from HSC72 cells in the absence of FANCA. A nonspecific band is indicated by an asterisk. (E) Immunoblot showing levels of FANCA and FAAP20 in cytosol and nuclear extracts of HSC93:shControl and HSC93:shFANCA cells; actin and ATR serve as controls. FANCA and FAAP20 are predominantly in the nuclear fraction, and FANCA knockdown results in reduced levels of FAAP20. (F) Immunoblot showing levels of FANCA and FAAP20 in total lysates of HeLa:vector and HeLa:shFANCA cells. Knockdown of FANCA results in decreased FAAP20. (G) Immunoblot of HeLa cell lysates showing that FAAP20 and FANCG, but not FANCL or FANCM, are induced by overexpression of FANCA. (H) Immunoblot showing levels of FANCA and FAAP20 in total lysates of HeLa:shControl and HeLa:shFANCA cells cultured in the presence (+) or absence (−) of Mg132 protease inhibitor. Inhibition of the proteasome pathway results in increased FAAP20 levels, despite low levels of FANCA. (I) Immunoblot showing core-complex proteins in input and IP samples from HSC72 cells stably expressing _His6-FLAGFANCA or _His6-FLAGFAAP20. FAAP20 induced on FANCA expression was able to coprecipitate with FANCA and FAAP20 failed to interact with other core-complex proteins in the absence of FANCA.