Fig. 2

Tyr724 residue of ELMO1 is a phosphorylation site by Src. a Schematic representations show ELMO1 protein with the tyrosine residues mutated (from tyrosine to phenylalanine residue, Y60F, Y216F, Y352F, Y356F, Y395F, Y511F, Y646F, Y662F, Y720F, Y724F, upper), and its deletion mutant (663–727 amino acids) fused with GST (lower). PH: Pleckstrin Homology domain, PxxP: proline-rich motif. b In vivo phosphorylation of ELMO1 mutants by Src. HEK293T cells were transfected with either pCMV-Myc-ELMO1 or its defective mutants on tyrosine, together with pCXN2-Flag-Src Y527F. Lysates from these cells were subjected to immunoprecipitation with anti-Myc Ab for ELMO1, followed by immunoblotting with Abs to PY (upper) and Myc (middle). Expression of extrinsic Src was also examined using anti-Flag Ab (lower). c In vivo phosphorylation of ELMO1 Y724F mutant by Src and Hck. Lysates from HEK293T cells expressing Flag-tagged ELMO1 and its Y724F mutant in the presence or absence of Src and Hck were subjected to immunoprecipitation and immunoblot analyses. d In vivo phosphorylation of ELMO1Δ662 by Src. HEK293T cells were transfected with pEBG-ELMO1Δ662 and its mutant forms (Y720F, Y724F, Y720/724 F), together with pMik-Src Y527F. Lysates from the cells were subjected to the pull-down assay with glutathione-sepharose beads, followed by immunoblotting with Abs to PY (top) and GST (middle). Expression levels of Src (intrinsic Src and its extrinsic Y527F) were examined using anti-Src Ab (bottom). GST-ELMO1Δ662: ELMO1Δ662 fused with GST