The carboxy-terminal SH3 domains of TKS5 interact with the PRD domain of FGD1. (A) Lysates of MDA-MB-231 cells expressing TKS5^GFP or not (Ctrl) were immunoprecipitated with GFP antibodies. Bound proteins were analyzed with TKS5 and FGD1 antibodies (immunoprecipitation, IP). 5% of total lysate was loaded as a control (input). Equal loading was controlled using GAPDH antibody. (B) Lysates of MDA-MB-231 cells expressing FGD1^GFP or not (Ctrl) were analyzed by immunoprecipitation as in A. (C) GST or indicated GST-FGD1 constructs on beads were incubated with MDA-MB-231 cell lysates. TKS5 protein was identified by immunoblotting analysis with TKS5 antibody. 5% of total lysates was loaded as a control (input). A schematic representation of FGD1 and GST constructs is shown. The endogenous TKS5 protein pulled down by GST-FGD1^18-335 represents ∼1.5% of the input. (D) Pull-down assays of indicated TKS5^GFP constructs from MDA-MB-231 cell lysates with GST-FGD1^18-335 protein immobilized on beads. Bound material was analyzed with GFP antibody. 5% of total lysates was loaded as a control (input). A schematic representation of the TKS5 constructs is shown on the upper part. The GFP-tagged TKS5 and TKS5_324-1118 proteins pulled down by GST-FGD1^18-335 represent 1.35% and 2.85% of the input, respectively. Molecular weights are in kD.