Generation of an affinity-purified rabbit polyclonal antibody against FAA that specifically detects FAA in Western blotting and immunoprecipitation experiments. (A) Western blot showing the approximately 37-kD His-FAA1 fusion protein in bacterial extracts and after purification using the affinity-purified antibody. The His-tagged fusion protein encompasses the first 321 amino-terminal residues of FAA and was used to raise the antibody. Lysates derived from noninduced bacteria or isopropyl-β-thiogalactopyranoside (IPTG)-induced bacteria are indicated with (−) or (+), respectively, and the purified protein with (P). (B) Western blot using extracts derived from normal cells or cells representing five different FA complementation groups. The FAA-specific band of approximately 163 kD is indicated. Blots were reprobed with an anti–β-tubulin antibody as a control for the amount of protein loaded per lane. Lymphoblastoid cell lines used represent the following FA subtypes: HSC93 and nl, normal cells; HSC72 and HSC99, FA-A; HSC230, FA-B; HSC536, FA-C; HSC62, FA-D; EUFA130, FA-E. HSC72+FAA indicates HSC72 cells stably transduced with a retroviral vector expressing FAA. HSC99+FAA indicate cells that were stably transfected with an episomal expression vector containing the FAA cDNA. (C) Immunoprecipitation of FAA from whole-cell extracts, generated in RIPA buffer, from metabolically labeled COS1 cells either mock-transfected (−) or transiently transfected with pcDNA-FAA5.5 (+).