FIGURE 2:

A single Pxl1 PxxP motif is necessary and sufficient for Cdc15C binding. (A) Schematic of Pxl1 with position of PxxP motifs indicated by asterisks. (B, C) In vitro binding assays with Cdc15C (residues 441–end) or Cdc15C1 (residues 600–end) and indicated MBP-Pxl1 variants. The binding reactions were analyzed by SDS–PAGE and Coomassie blue staining. (D) In vitro binding assays with biotin-labeled Pxl1 peptides and GST-Cdc15C1, GST-Cdc15C1(W903S), GST-Cdc15-SH3, or GST-Cdc15-SH3(W903S). Proteins bound to the streptavidin resin were analyzed by SDS–PAGE followed by immunoblotting. (E) Binding curves for Pxl1 peptide (aa 177–188) binding to Cdc15C1. The dissociation constant, calculated from three experiments is indicated. Error bars represent SEM.