Fig. 2.

(A) ESDN domain structure and corresponding human amino acids including the seven intracellular tyrosines found in YxxP motifs. TM=transmembrane. (B) Weblogo of the Crk-SH2 binding motif aligned with the ESDN intracellular YxxP motif sequences in descending order (high, medium and low) of their Scansite predicted Crk-SH2 binding. Note putative phosphotyrosine residues are at position zero. (C) Multiple regulated phosphotyrosine residues participate in the binding of ESDN to the CrkL-SH2 domain. HEK 293 cells transfected with the indicated constructs were treated as indicated. Whole cell extracts were subjected to SDS-PAGE and immunoblotting directly with the indicated antibodies, or after being subjected to either GST-CrkL-SH2 pulldowns (upper panels) or anti-Flag immunoprecipitations (lower panels). The Ponceau stain of the membrane prior to immunoblotting indicates the levels of the transferred GST-CrkL-SH2 in each lane. *Indicates non-specific background bands.