<p>(A and B) Tandem mass spectrometry (MS/MS) spectra of peptides (A) ELSEYNATAIK and (B) AGAIYVFQR are shown. Fragments critical for localization of phosphorylation sites are labeled in red. (C) Schematic of the known functional domains of EphB2 receptor. LBD, ligand-binding domain (purple); Cys, cysteine-rich domain (white); FN3, fibronectin type III repeat domain (orange [N-terminal FN3 (nFN3)]and blue [cFN3]); TM, transmembrane domain; JM, juxtamembrane domain (yellow); TK, tyrosine kinase domain (red), SAM (grey), sterile α-motif; PDZ, PSD-95/DLG1/ZO-1 domain (green). (D) Alignment of cFN3 domains of EphB2 with Eph family proteins (Uniprot database) using ClustalW2 software. EphB2 Y504 (red) corresponds to a conserved tyrosine residue, whereas Y481 (blue) is only conserved in mammals (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002457#pbio.2002457.s001" target="_blank">S1E Fig</a>, grey). Yellow indicates identical amino acids with mouse EphB2. (E) Alignment of cFN3 domain of EphB2 with other FN3-containing molecules. Y504 and neighboring residues in identified peptide (red) are well conserved amongst Eph family proteins (55.4% identical amino acids) or FN3-containing molecules (45.5% identical amino acids), whereas Y481 and neighboring residues in identified peptide (blue) are less well conserved amongst species (19.6% and 11.6% identical amino acids for Eph family proteins and FN3-containing molecules, respectively). (F) Top blot shows HEK293T lysates probed with a phospho-specific antibody generated against EphB2 Y504 (α-EphB2 p*Y504). Middle blot shows same lysates probed for EphB2. Bottom blot shows lysates probed for EphB2 p*Y662 (EphB2 kinase activity). Lanes were loaded with lysates of HEK293T cells transfected with full-length (FL) EphB2 wild type (Y) or Y504F (F), kinase-dead (KD) EphB2 wild type (Y) or Y504F (F), and truncated (TR) EphB2 wild type (Y) or Y504F (F). (G) Left blots show synaptosome lysates prepared from wild-type (WT) CD1 mouse brain and right blots show synaptosome lysates prepared from the spinal cord. Gels were loaded with nonsynaptic (S1), crude synaptosomal (P2), and synaptosomal (Syn) fractions. The top 2 blots were probed with α-EphB2 p*Y504 antibody before (upper) and after (lower) incubation with calf intestinal alkaline phosphatase (CIP) overnight to remove phosphate groups. The third and fourth blots were probed with α-EphB2 before and after CIP treatment. The fifth blot was probed with α-GluN1. The bottom blot was probed with α-PSD-95. (H) Blots show synaptosome lysates prepared from WT CD1 mouse brain at P9, P15, or P21. The top blot was probed with α-EphB2 p8Y504 antibody, the second blot was probed with α-EphB2, the third blot was probed with α-GluN1, the fourth blot was probed with α-GluN2B, and the fifth blot was probed with α-PSD-95. The bottom blot was probed with α-GAPDH as a loading control.</p
