EphB1 phosphorylates Caskin1 on tyrosine 296 and 336 (1) . (a) V5-Caskin1 and HA-EphB1 were transiently coexpressed in COS7 cells. Cell lysates were then subjected to immunoprecipitation with anti-V5 antibody. Bound proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-phosphotyrosine and anti-V5 antibodies. (b) V5-Caskin1 and HA-EphB1 were transiently coexpressed in COS7 cells as in Figure4a, however, after immunoprecipitation with anti-V5 antibody protein samples were stained by Coomassie blue. The position of the bands sent to mass spectrometry analysis (Caskin1) is indicated. (c) The 3D structure of the rat Caskin1 SH3 domain was modeled using the public I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER) structure prediction server. The positions of the two tyrosines phosphorylated by EphB1 are indicated.