Fig. 3: 643943 releases CASP7 from the inactive complex with XIAP.
A In vitro binding assay for GST-XIAP interaction with CASP7 in the presence of increasing 643943 concentrations (0.1 and 0.5 mM). GST-XIAP was pulled down using Glutathione beads and CASP7 was detected via Western blot. Like I-Lys, 643943 disrupted the GST-XIAP:CASP7, but not GST-XIAP:CASP3 complexes. B Activity levels of CASP7 complexed with GST-linker-BIR2 and subsequently treated with 100 μM of different agents including 643943, 119, a peptide AVPFVASLPN derived from SMAC, and a mutant peptide. C Fluorescence spectra of CASP7 in the absence (black) and presence of different concentrations (1, 3, 5, 10, and 15 μM) of 643943 as shown in orange, red, blue, green, and purple, respectively (left panel). The fluorescence spectrum (gray) of 643943 has a λmax of 420 nm, so that the addition of 643943 did not contribute to the fluorescence changes at 346 nm, the λmax of CASP7. The right panel shows the changes of fluorescence at 346 nm during titration. D BIAcore traces showing the binding of 0.28 μM GST-linker-BIR2 in the absence (black) and presence of different concentrations of 643943 as shown in orange, yellow, green, cyan, blue, and pink, respectively (left panel) and 119 as a negative control (right panel). The concentrations of 643943 and 119 used were 0, 0.5, 10, 15, 20, 30, and 40 µM. A–D Data were obtained from three independent experiments.
