FIG. 2.

BIR2 of XIAP binds AIF; mature AIF begins at residue 55. (A) HEK 293 cells were transfected with plasmids encoding various XIAP domain truncations in fusion with GST along with a plasmid encoding full-length human AIF. Cell lysates were prepared, and XIAP was precipitated with glutathione (GSH) beads. The presence of AIF in precipitated complexes was determined by immunoblotting. (B) Full-length human AIF-TAP was expressed in HEK 293 cells. AIF was precipitated from cell lysates using IgG-Sepharose beads; eluted proteins were then separated by SDS-PAGE and transferred to PVDF membranes. Following Coomassie blue staining, the AIF band was excised and subjected to Edman degradation in order to determine the mature amino terminus. Numbers at left of panels A and B are molecular masses in kilodaltons. (C) Primary sequence of full-length human AIF. The underlined sequence is one of four AIF peptides recovered from analysis of XIAP-TAP. The gray arrow is the first residue of mature AIF as reported by reference 51; the black arrow is the first residue of mature human AIF as determined by this study.