Fig. 4. Semaphorin 3A activates myosin II. (A) Neurons were loaded with 2.5 µM of the volumetric fluorescent reporter CellTracker prior to treatment with semaphorin 3A or BSA (control) for 10 minutes. Cultures were then fixed and stained with an antibody against phosphorylated regulatory myosin light chains (rMLC-p). Treatment with semaphorin 3A increased the levels of rMLC-p in axons relative to controls. (B) Quantification of the ratio of rMLC-p staining to CellTracker staining (sample size is given within bars). Semaphorin 3A increased rMLC-p staining in the distal 20 µm of the axon by 69% (P<0.0001, Welch t-test). The mean and s.e.m. are shown in all presentations of quantitative data. (C) Pretreatment with 10 µM y-27632 (30 minutes) prevents the semaphorin-3A-induced increase in the ratio of rMLCp to CellTracker staining (P>0.7, Welch t-test; sample site is givin within bars). Data are shown normalized to treatment with y-27632 alone.