Figure 1From: Complex formation of EphB1/Nck/Caskin1 leads to tyrosine phosphorylation and structural changes of the Caskin1 SH3 domainNck interacts with Caskin1 through its SH3 domains. (a) Endogenous Caskin1 was immunoprecipitated (IP) from rat brain lysate with a monoclonal anti-Caskin1 antibody. After SDS-PAGE and transfer to nitrocellulose, samples were analyzed by anti-Nck and anti-Caskin1 antibodies. (b) V5 epitope-tagged Caskin1 and/or GFP-tagged Nck were transiently expressed in COS7 cells, and then cell lysates were immunoprecipitated with an anti-V5 antibody. Immunoprecipitates were immunoblotted with anti-GFP and anti-V5 antibodies. (c) Full length Nck, the three SH3 domains together (Nck-SH3-all), and individual SH3 domains (SH3/1, SH3/2 and SH3/3) of Nck were generated as GST fusion proteins. These fusion proteins were immobilized on glutathione-agarose beads and used for protein precipitations from lysates of COS7 cells expressing V5-Caskin1. Precipitated proteins were separated on SDS-PAGE and transferred to nitrocellulose, and then samples were immunoblotted with anti-V5 antibody. (d) Coomassie stained gels with GST fusion proteins used in the previous experiment. These results are representative of three experiments.Back to article page