Identification of proteins associated with the PDZ-binding motifs of Amot, AmotL1, and AmotL2. (A) Peptides corresponding to the last 19 C-terminal amino acids of Amot, AmotL1, and AmotL2 including the PDZ-binding domains were used for pull-down experiments of mouse aortic endothelial lysate. Peptides lacking the 3 last amino acids were used as negative controls. (B) Proteins that bound to peptides were separated by SDS-PAGE and visualized by silver staining and analyzed by mass spectrometry. Data regarding peptide coverage can be found in Table S1. (C) The table summarizes the proteins pulled down with the full-length peptides but not with the truncated peptides. Patj was also found to associate with Amot by yeast 2-hybrid screening as described in “Yeast cotransformation assays.” (D) Myc-Pals1, myc-Patj, or His-Syx were cotransfected in HEK293 cells together with human p80 Amot or p80 ΔYLI (negative control lacking the PDZ-binding motif). Protein-protein interaction was verified by coimmunoprecipitation analysis. Endogenous Mupp1 was immunoprecipitated with transfected human p80Amot (top left panel). The vertical lines indicate repositioned gel lanes.