FIGURE 6.

S100 proteins activate PP5 and increase the dephosphorylation of Tau in vitro and in vivo. A, pSer-396 or pSer-409 Tau was incubated with His-PP5 and S100A1 (lanes 4 and 5), S100A2 (lanes 6 and 7), S100B (lanes 8 and 9), or S100A12 (lanes 10 and 11) in the presence of 1 mm CaCl₂ or EGTA for 10 min at 37 °C. Samples were separated with SDS-PAGE and analyzed by Western blotting (WB) with anti-pSer-396 Tau, anti-pSer-409 Tau, or anti-Tau antibody. A detailed description can be found in the experimental procedures. Lane 1, non phosphorylated TAU; lanes 2-11, phosphorylated Tau; lane 3: PP5 treatment only. B, COS-7 cells stably expressing Tau were either transfected with pME18S vector only (Mock) or S100A1 expression vector (S100A1) or not transfected (Control). After 48 h, cells were stimulated with 5 μm ionomycin for 15 min or 60 min. Lysates were prepared and analyzed by Western blotting with anti-pSer-396 Tau, anti-Tau, anti-S100A1, or anti-PP5 antibody. C, the density of each band obtained using anti-pSer-396 Tau and anti-Tau antibody was measured by Image J software (National Institutes of Health) and the % ratio of pSer-396 Tau against total Tau was calculated. Each column represents the mean ± S.D. calculated from three to five independent experiments.