A5795

IP assays and Western blottings were carried out as previously described (Laporte et al., 2011 (link); Lee and Wu, 2012 (link); Wang et al., 2014 (link)) with the following modifications: 1) 200 mg (Figure 2C) and 40 mg (Figure 9C) of lyophilized cells for each strain were used for IPs and Western blottings, respectively, due to the low abundance of Gef3; 2) The monoclonal anti-Myc antibody (1:1000 dilution; 9E10; Santa Cruz Biotechnology, Santa Cruz, CA) was used to detect Gef3-13Myc. Rho GTPases were detected by monoclonal anti-GST antibody (3G10/1B3, 1:10,000 dilution; NB600-446; Novus Biologicals, Littleton, CO). A monoclonal antibody against FLAG (1:2000 dilution; F1804; Sigma-Aldrich) was used to detect 3FLAG-Gef3, and 6His-Gef3 was detected by anti-His antibody (1:10,000 dilution; 631212; Clontech, Mountain View, CA). Secondary anti-mouse antibody was used at 1:10,000 dilution for all the primary antibodies mentioned. In addition, triple-hemagglutinin (HA)–tagged wt and mutant Rho4 were detected by anti-HA antibody (1:2000 dilution; 3F10; Roche, Mannheim, Germany), and a secondary anti-rat antibody (A5795; Sigma-Aldrich) was used at 1:4000 dilution.