Figure 2

The effect of plexinB1 mutations on Rac signalling. A). Mutation of plexinB1 impairs inhibition of Pak1 phosphorylation. The phosphorylation status of Pak1 in COS-7 cells transfected with plexinB1 (WT or mutant), RacL61 and Pak1 was monitored by western blotting using anti- phospho199/204-Pak1 antibody. B). Mutation of plexinB1 impairs inhibition of cell spreading. COS-7 cells transfected with RacL61 and plexinB1 (WT or mutants) were plated on fibronectin for 2 h and those with a large surface area and discernible lamellipodia ('spread' phenotype) scored. Bars represent means +/- SE, *p < 0.05 versus RacL61 + WT, one tail t-test. C). Mutation in plexinB1 inhibits Rac-dependent trafficking of plexinB1 to the cell surface. COS-7 cells co-transfected with plexinB1 (WT or mutant) and RacL61 were treated with Sema4D-AP fusion protein and cell surface binding of Sema4D-AP assessed. i). Sema4D-AP cell surface binding detected with BCIP/NBT solution, representative of 3 independent experiments, ii). Western blot to show expression of VSV-plexinB1 and myc-RacL61 in transfected cells. iii). Bound alkaline phosphatase activity in transfected cells. Bars represent means +/- SE. *p < 0.01 versus vector without RacL61; **p < 0.01 versus WT with RacL61, one tail t-test. D). Mutation of plexinB1 does not affect Sema4D binding. HEK293T cells were cotransfected with VSV-plexinB1 (WT or mutant) and Sema4D-Fc. The plexinB1 protein in the cell lysate was immunoprecipitated with anti-Fc antibody.