Fig. 4

Slit2 inhibits Cdc42 activity through srGAP1. a Purification of GST-PAK1 protein. GST-PAK1 fusion proteins were expressed in E. coli (BL21 strain) and purified using GST-sepharose beads. M: protein marker; Total protein from control BL21 strain(1) and BL21 strain expressing GST-PAK1(2); 3: Purified GST-PAK1 protein. b Slit2 inhibited Cdc42 activity in a Robo-dependent manner. Ectopic expression of Slit2 inhibited Cdc42 activity (Cdc42-GTP) in LoVo cells, which could be blocked by ectopic expression of RoboN. GTP-loading of Cdc42 (GTP-Cdc42) in LoVo cell extracts was pulled down using GST-sepharose beads loaded with GST-PAK1 protein and was detected by Western blot using an anti-Cdc42 antibody. c Slit2 inhibited Cdc42 activity through srGAP1. Ectopic expression of srGAP1 enhanced the inhibitory effect of Slit2 on the GTPase of Cdc42, whereas blocking srGAP1 activity by srGAP1ΔGAP rescued the Cdc42-GTP levels in Slit2-overexpressed LoVo cells