In vivo phosphorylation of FANCA protein in various cells and patient-derived mutant FANCA proteins.
(A) In vivo phosphorylation of FANCA in normal lymphocytes. Lymphocytes from cord blood (CB Ly; lanes 1 and 2) and peripheral blood (PB Ly; lane 3), and lymphoblastic cells (LCL; lane 4) from a normal control were metabolically labeled with 32P, lysed, and subjected to immunoprecipitation (IP) with preimmune serum (Pre; lane 1) or anti-FANCA antibody (lanes 2-4). Western blots of FANCA are shown at the bottom. (B) In vivo phosphorylation of FANCA in GM6914/wt-FANCA, HeLa, Jurkat, and Mo-7e cells. Western blots of FANCA are shown at the bottom. (C) MMC sensitivities of GM6914 cells transduced with wt-FANCA, FANCA(Arg435Cys), and FANCA(1239-43del). Cells were seeded in the presence of various concentrations of MMC and cell survival was assayed 4 days after seeding. (D) Monoubiquitination of FANCD2 in GM6914 cells transduced with wt-FANCA, FANCA(His1110Pro), FANCA(Arg1117Gly), FANCA(Arg435Cys), and FANCA(1239-43del). (E) In vivo phosphorylation of wt-FANCA, FANCA(His1110Pro), FANCA(Arg1117Gly), FANCA(Arg435Cys), and FANCA(1239-43del) expressed in GM6914 cells. Western blots of FANCA are shown at the bottom.
