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. 2021 Jan 15;7(3):eabc8590. doi: 10.1126/sciadv.abc8590

Fig. 2. Free Nt-α-amino peptides of cytosolic proteins bind to IAPs’ BIR domains in vitro.

Fig. 2

(A) Table with examples of known IAP antagonists [DIABLO, HTRA2, and dr Reaper (rpr)], IAP binders (CASP9), and cytosolic proteins with potential N-terminal IBM motifs (COMMD10, NIT1 Isoform1, and CDC20). Gene name, (potential) IBM with N-terminal amino acid in processed protein, cellular localization, and function are shown. (B) Fluorescein isothiocyanate (FITC)–labeled peptides (free Nt-α-amino, indicated as Nt-free, or Nt-Ac, each used at a concentration of 20 nM) of DIABLO (left), CASP9 (middle), and COMMD10 (right) were incubated for 30 min with increasing concentrations of His-tag BIR domain constructs (XIAPLinker-BIR2-BIR3, BIRC3BIR3, or XIAPBIR1). Fluorescence polarization (FP) was measured at excitation and emission wavelength of 470 and 525 nm and data (millipolarization units, mP) were plotted as a function of BIR domain concentration and fitted with a logistic fit using Origin7.0. Kd values are summarized in table S3. Measured combinations are shown schematically in the top panel.