Figure 1.

Hip1R interacts with the SH3 domain of cortactin. (A) co-immunoprecipitation of Hip1R with cortactin from a clathrin coat extract. Cortactin immunoprecipitates (IP) and controls lacking antibody (C) were blotted with anti-Hip1R and anti-cortactin antibodies. (B) His-tagged Hip1R (40 nM) pull-down assays using GST and GST-cortactin 1–546; 1–81; 1–326; 324–546; 491–546 (10 μM). Bound (P) and unbound (S) fractions were analyzed by Western blotting (WB) using an anti-His tag antibody (upper panel) and by Coomassie blue staining (lower panel). (C) His-tagged Hip1R (1 nM) pull-down assays using the indicated concentrations of GST-cortactin. Total input (T) and bound fractions were probed with an anti-His tag antibody. The results were quantified to obtain the binding curve. The best fit of the curve gave a K_d=20 nM. (D) Summary of the binding assay results. All the experiments were performed three times with similar results.