Nck 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.