Figure 3.

The WXXXN motif in SmgGDS is important for binding of RAC1 and RAC1B but not RHOA or KRAS. HEK293T cells were transfected with HA vector, SmgGDS-558, or SmgGDS-558-AIPSA-HA, along with the indicated myc-tagged DN small GTPase. After 24 h, the cells were lysed and a portion of each lysate was saved for the total cell lysate. The remaining lysate was immunoprecipitated with the HA antibody, followed by ECL immunoblotting using HA and myc antibodies. Results are representative of three independent experiments. Densitometry of the proteins detected in the immunoblots is shown in Fig. S2. DN, dominant negative; ECL, enhanced chemiluminescence.