Figure 3.

Regulation of CDC42 by SRGAP1 and its mutants. A, Schematic drawing indicating the mutation positions in the SRGAP1 protein. The domain regions are shown based on National Center for Biotechnology Information reference sequence NP_065813.1. B, SW1736 cells were cotransfected with HA-ROBO1 and with either empty-MYC vector or MYC-SRGAP1 wild-type (wt) or mutants (Q149H, A275T, R619C). After 48 hours, cells were either left untreated (−) or were treated with conditioned medium from HEK 293 cells overexpressing HIS-SLIT2 (+). GST fusion proteins of PBD-conjugated beads were used to pull down GTP-bound forms of the CDC42 GTPase. After GST pulldown (PD), CDC42 GTPase was detected using anti-CDC42 antibodies. Whole-cell lysates were also immunoblotted with anti-MYC, anti-HA, or anti-CDC42 antibody (input controls). C, Data from the experiments above were quantified using ImageJ software (http://rsb.info.nih.gov/ij/).