(A) Immunohistochemistry demonstrates that Akt and ERK are expressed in vaginal epithelium of both WT (Sema4D+/+) and Sema4D−/− mice at 5 weeks old at the time of vaginal opening. (B) Western blots show the expression patterns of Akt, ERK, and the respective phosphorylated forms during postnatal vaginal development in WT mice. Both pAkt and pERK levels are lower in samples from 5-week-old mice than in samples from any other stages of development. Age (weeks): vaginal protein extracts from 2-, 3-, 4-, 4.5-, 5-, 8-, 9-, or 18-week-old mice. Each value represents the mean ± SEM of 6 mice. *P<0.05, ANOVA. (C) Western blot analysis reveals significantly higher expression of pAkt and pERK in vaginal tissue samples from 5-week-old Sema4D−/− mice than in vaginal tissue samples from 5-week-old WT (Sema4D+/+) mice. The ratios of pAkt to Akt and separately of pERK to ERK were higher in vaginal tissue samples from 5-week-old Sema4D−/− than in vaginal tissue samples from WT mice. Data are shown as the mean ± SEM; n = 6 per mouse group. *P<0.05, Student’s t-test.