Figure 3.
Analysis of Fgd5-Rho GTPase interaction. (a and b) Direct binding of Fgd5 to Rac1. Incubation of 5 μM Fgd5-DH-PH-FYVE triple domain (a), or the Fgd5-DH-PH double domain (b) with immobilized GST or GST-tagged RhoA, Cdc42 or Rac1. Arrows indicate the Fgd5 bands, while the GST probes can be seen in the background. (c) The Fgd5 DH-PHF803K double domain mutant does not bind Rac1. (d) Co-immunoprecipitation of Fgd5 and Rac1. Lysates (left panels) from HEK293T cells expressing GFP, GFP-Fgd5 (full length), GFP-Fgd5∆DH (deletion of the DH domain) were subjected to immunoprecipitation with GFP antibodies (right panels). Upper panels were immunoblotted with anti-GFP, * indicates GPF proteins in lysates. Lower panels were immunoblotted with anti-Rac1, showing equivalent levels of Rac1 in lysates (left) and its detection only in the GFP-Fgd5 immunoprecipitation (right). HC and LC indicates heavy and light chain IgG in the immunoprecipitate