ONHAs pretreated with 25 μM estrogen were exposed to tBHP to induce oxidative stress. (A) Phosphorylation of Tau at Ser422 was examined by immunoblotting. ONHAs showed Ser422 tau phosphorylation under control conditions (see mock). (B) tBHP treated ONHAs had a significant reduction in tau phosphorylation versus mock (**, p=0.0027). ONHAs pretreated with estrogen for 2 h followed by tBHP treatment had a significant increase in phosphorylated tau versus ONHAs treated with tBHP alone (*, p=0.0482). When estrogen pretreatment was increased to 18 h tau phosphorylation was increased significantly when compared to tBHP treatment (**, p=0.0037). Estrogen pretreatment for 18 h was more effective than a 2 h pretreatment (**, p=0.0085) in restoring phosphorylation of tau at Ser422. ONHAs pretreated with estrogen for 18 h (ns, p=0.5598) followed by tBHP treatment showed no significant difference from mock treated ONHAs, while the 2h pretreatment still showed a significant difference compared to mock treated (**, p=0.0070). Experiments were done in triplicate (n=3) and values were depicted as mean +/− SEM and analyzed using ANOVA (p=0.0002). For statistical comparison, the Bonferroni post-hoc test and Student t-test were used. Statistical significance was set at p ≤ 0.05.