Fig. 6: The assembly of the export complex is disturbed by unmodified and peptide-linked tweezer molecules.
From: Specific inhibition of the Survivin–CRM1 interaction by peptide-modified molecular tweezers
a 293T cell lysate with overexpressed Survivin142-HA was preincubated with either unmodified tweezer (TW), TW-ELTL, TW-ELTLGEFL, or a scrambled peptide-modified tweezer, TW-LFEEGLLT, at concentrations ranging from 0.01 to 200 µM. GST-CRM1 was mixed with either non- or preincubated cell lysates in the presence of recombinant RanQ69L and dGTP to allow complex assembly. GST-CRM1 and interacting proteins were pulled by GSH-Sepharose beads. Proteins in input and beads samples were analyzed via immunoblotting with antibodies specific for GST or HA. For each tweezer, samples derive from the same experiment and gels/blots were processed in parallel. Direct comparison for this exact concentration range was performed once for TW, TW-ELTL, and TW-ELTLGEFL and for TW-LFEEGLLT in three technical replicates. b, c Atto488-labeled Survivin120 was preincubated with CRM1_1-1062VLV430AAA mutant in a ratio of 1:5 and titrated with supramolecular tweezers up to approx. 180 µM. Fluorescence anisotropy was measured (n = 1) (b), and IC50 values were determined from the resulting curves (c). TW light blue/ triangles, TW-ELTL blue/squares, TW-ELTLGEFL dark blue/circles. Source data are provided as a Source Data file.
