Figure 4

WWOX suppresses Tau phosphorylation. (a) GST, GST–WWOX or GST–WWOX L404A (0.2 μg) were incubated with 30 ng His-GSK3β for 30 min in a reaction buffer and incubated with 0.5 μg Tau protein, ATP cocktail, and 10 Ci of [γ-³²P] ATP for 10 min at 20 °C. The reaction was stopped by adding 2 × sample buffer and heating at 100 °C for 10 min. This was followed by SDS-PAGE, and phosphorylation signals were evaluated using autoradiography. (b) WWOX (1, 3 and 10 μg) inhibited Tau phosphorylation by GSK3β in a dose-dependent manner. GST–WWOX was pre-incubated with His-GSK3β for 30 min in a reaction buffer including optimised quantities of ATP and Tau protein. The reactions were then subjected to western blot analysis of Tau, pTauS396 and S404. (c) SH-SY5Y cells were transiently transfected with GFP, GFP–WWOX or GFP–WWOX L404A. Cells were lysed, and a western blot analysis was carried out using antibodies against pTauS396, pTauS404, pTauS422, Tau and actin. Actin expression was evaluated to verify equal loading, and GFP expression was evaluated to confirm the protein expression level. (d) A scrambled peptide and WWOXtide_388−407 were each incubated with 30 ng His-GSK3β, ATP-cocktail, and 10 Ci of [γ-³²P] ATP for 10 min at 20 °C. The reaction was stopped by adding 2 × sample buffer and was followed by SDS-PAGE. Phosphorylation was detected with autoradiography