Fig. 3. Site-specific effect of LiCl and Bu1 on tau phosphorylation in differentiated PC12 cells. PC12 cells were differentiated with FGF-2 for 7 days and then treated with medium alone (C), or medium containing 20 mM LiCl (L) or 10 µM Bu1 (B). (A) The cell lysates (20 µg/lane) were analyzed by ¹²⁵I western blotting with phospho-independent antibody R134d to tau and antibodies to different phosphorylation sites on tau (indicated at the bottom of each individual panel). Bars on the left indicate the positions of 70 kDa and 50 kDa molecular mass markers. (B) Quantitative evaluation of the effect of the inhibition of GSK-3 with LiCl (white bars) and of cdk5 with Bu1 (hatched bars) on the phosphorylation of tau at different sites. The values represent the degrees of inhibition of site specific phosphorylation calculated after normalization of each individual value with the corresponding values for total tau (R134d). Values are means of three to nine individual western blots. Bars represent s.e.m.; ^*P<0.05, ^**P<0.01, ^***P<0.001, Student's t-test. (C,a) Western blot analysis of tau bound (B) or unbound (U) to microtubules in differentiated PC12 cells (NGF, for 4 days) pretreated with (Li+) or without (Li–) 20 mM LiCl for 3 hours. Blots were untreated (Tau-1) or treated with alkaline phosphatase (Tau-1 dp) and stained with Tau-1 or directly stained with DM1A for -tubulin (data not shown). Lines on the right side of the western blots indicate the positions of the 70 kDa and 50 kDa molecular mass markers. (b) Quantitative analysis of the degree of phosphorylation of tau at the Tau-1 site from a in the bound fraction. Lithium (Li+) markedly decreased tau phosphorylation at the Tau-1 site. (c) Quantitative analysis of the ratio of bound tau to unbound tau (B/U) from a (mean±s.e.m.). Lithium (Li+) did not affect tau's ability to bind to microtubules (compare Li- with Li+).