Figure 6.

Coexpression of MARK4^ΔG316E317D causes accumulation of total tau and tau-phosphorylated at sites other than Ser-262/356 in vivo. A, phosphorylation profile of tau expressed alone or coexpressed with MARK4^wt or MARK4^ΔG316E317D in vivo. Fly head extracts were separated by Phos-tag SDS-PAGE, followed by Western blotting with anti-tau antibody. Coexpression of MARK4^wt increased the abundance of faster-migrating tau. By contrast, coexpression of MARK4^ΔG316E317D increased the abundance of slower-migrating tau. B, Western blotting was performed using phospho-specific antibodies for SP/TP sites such as AT8 (pS202/T205), anti-pThr231 (pT231), and PHF1 (pS396/404), as well as pan-tau antibody T46 (tau). Actin was used as a loading control. Representative blots (top) and quantification (bottom panels, normalized either to actin or to total tau) are shown. Means ± S.D. (error bars); n = 4. n.s. (not significant), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.005 (one-way ANOVA and Tukey's post-hoc test). C, MARK4^wt and MARK4^ΔG316E317D phosphorylated tau in the same pattern in vitro. MARK4^wt and MARK4^ΔG316E317D expressed in HEK293 cells were immunoprecipitated and incubated with recombinant tau. Tau proteins were separated by Phos-tag SDS-PAGE, followed by Western blotting with anti-tau antibody T46.