Figure 2.

(A) Cytoplasmic tail sequence alignment compares ephrinB2 with integrins ?1 and ?3 (upper panel). Membrane-proximal NPxY motif in integrins ?1 and ?3 is bolded. Serine/threonine residues are bolded and underlined. NxxY motif is bolded and boxed, and ephrinB2 PDZ-binding motif is underlined. Conservation of ephrinB2 Ser/Thr residue and NxxY motif in human, mouse, and zebrafish was examined using Clustal Omega program (http://www.ebi.ac.uk/Tools/msa/clustalo/) (lower panel). ?*? indicates fully conserved amino acid residue. (B) Pulldown assay using purified his/Avi-tagged cytoplasmic tails of ephrinB2 WT, kindlin2-binding mutant 4A (NxxY to four alanine mutants), and integrin ?1A or ?IIb immobilized on NeutrAvidin beads was performed with a cell lysate expressing GFP-kindlin2. Bound proteins were washed and separated on SDS?PAGE, and binding of kindlin2 was detected by Western blot with anti-GFP antibody. Immobilized cytoplasmic tails were visualized by Coomassie brilliant blue staining. Both WT and 4A mutant were expressed equally well. (C) Pulldown assay of his/Avi-tagged cytoplasmic tails of ephrinB2 WT, 4A, or integrin ?IIb immobilized on NeutrAvidin beads with a cell lysate expressing GFP-Dvl2. Associated proteins were subjected to SDS?PAGE and subsequent Western blotting. Dvl2 was recognized by an anti-GFP antibody. Immobilized cytoplasmic tails were stained by Coomassie brilliant blue. (D) In vitro binding assay mapping the kindlin2-binding site on the ephrinB2 cytoplasmic tail. Purified kindlin2 was mixed with cytoplasmic tails of ephrinB2 WT, kindlin2-binding mutant (4A), or integrin ?IIb immobilized on NeutrAvidin beads and assessed for binding. Anti-kindlin2 antibody was used to detect bound kindlin2 in Western blot, and cytoplasmic tails were stained by Coomassie brilliant blue. (E) In vitro binding assay between purified kindlin2 and cytoplasmic tails of ephrinB2 with mutations of critical residues in NxxY motif and upstream serine residue immobilized on NeutrAvidin beads (left panel). The mutated residues were indicated in the ephrinB2 cytoplasmic tail sequence (right panel). Bound kindlin2 was recognized by anti-kindlin2 antibody in Western blot, and cytoplasmic tails were detected by Coomassie brilliant blue. (F) Binding of kindlin2 was quantified by densitometry, normalized to cytoplasmic tail loading, and then calculated relative to the binding of ephrinB2 WT (means ± SEM; n = 3). (G) Pulldown analysis of his/Avi-tagged cytoplasmic tails of ephrinB2 WT or 4A as a negative control, immobilized on NeutrAvidin beads with a cell lysate expressing GFP-kindlin2 WT or W615A, respectively. Bound proteins were fractionated on SDS?PAGE and probed with an anti-GFP antibody in Western blot. Immobilized cytoplasmic tails were visualized by Coomassie brilliant blue staining. (H) Pulldown of his/Avi-tagged cytoplasmic tails of integrin ?1A or ?IIb on NeutrAvidin beads with a cell lysate expressing GFP-kindlin2 WT or W615A, respectively. Pulldown proteins were washed and separated on SDS?PAGE. GFP-kindlin2 was recognized by anti-GFP antibody in Western blot. Purified cytoplasmic tails on NeutrAvidin beads were visualized by Coomassie brilliant blue staining. (I) His/Avi-tagged cytoplasmic tails of ephrinB1, ephrinB3, and integrin ?1 or ?IIb immobilized on NeutrAvidin beads were mixed with a cell lysate expressing GFP-kindlin2. The pulldown results were visualized by SDS?PAGE and Western blotting with anti-GFP antibody. All mutants were expressed equally well as WT. (J) Protein alignment (right panel) of cytoplasmic domains ephrinB1, ephrinB2, and ephrinB3 was generated by Clustal Omega. ?*? denotes fully conserved amino acid residue, and ?:? indicates conserved residue with similar properties among ephrinB cytoplasmic tails. (K) Pulldown of kindlin3 with his/Avi-tagged cytoplasmic tails of ephrinB2 WT, 4A, and integrin ?1A or ?IIb immobilized on NeutrAvidin beads. Bound proteins were separated on SDS?PAGE. Cytoplasmic tails were stained by Coomassie brilliant blue staining, and kindlin3 was probed by anti-GFP antibody in Western blot.

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