Transfection of COS7 cells with TM-agrin-GFP results in strong activation of Cdc42 and Rac1 but moderate inhibition of RhoA. Cells were co-transfected with wild- type Rho-family GTPases and GFP (control), or TM-agrin-GFP and the wild-type GTPases, or agrin, wild-type GTPases and dominant negative GTPases. After 24 h, cells were lysed and activation of Rac1, Cdc42 or RhoA was measured with a pull-down assay. Over-expressed Cdc42, Rac1 and RhoA were visualized by Western blotting using an anti-AU5 antibody (A–F). Each graph shows mean activation and standard error (n=3 experiments). (G) TM-agrin-GFP in the cell membrane and BRFs first appear 12 h after co-transfection with TM-agrin-GFP, at the same time that activation of Cdc42 is first detected (A).