Fig. 2. Differentiation of PC12 cells with NGF or FGF-2 upregulates the expression of tau and GSK-3ß and phosphorylation of tau at the Tau-1 site. (A) ¹²⁵I western blots of tau and GSK-3ß in PC12 cells differentiated with NGF or FGF-2; 12 µg of total cell lysate was applied per lane for SDS-PAGE. For immunostaining of tau with Tau-1 antibody, the blots were untreated (Tau-1) or pretreated with alkaline phosphatase (Tau-1 dp). Arrowhead indicates the position of high molecular mass tau; bars on the right indicate the positions of 70 kDa and 50 kDa molecular mass markers (from top). For GSK-3ß immunostaining, the blots were developed with antibody 127d to total GSK-3ß and with antibody Y216 to GSK-3ß phosphorylated at Tyr 216. (B) NGF and FGF-2 upregulate GSK-3ß activity in differentiated PC12 cells. GSK-3ß activities were measured by using 127d-immunoprecipitates of the extracts of cells cultured in NGF or FGF-2 for the indicated time periods. The activities (%) were converted based on the value of unstimulated cells as 100% (mean±s.e.m.). (C) NGF and FGF-2 increase tau phosphorylation at the Tau-1 site. The percentage of phosphorylation at the Tau-1 site was determined as described in Materials and Methods (mean±s.e.m.). (D) Lithium and valproate (VA) inhibit tau phosphorylation at the Tau-1 site in differentiated PC12 cells. PC12 cells differentiated by NGF for 4 days were further incubated without (control, C) or with LiCl (20 mM, 3 hours, LiCl), butyrolactone 1 (10 µM, 6 hours, Bu1), PD98059 (50 µg/ml, 1 hour, PD) or VA (0.6 mM, 3 hours, VA), lysed and analyzed by western blots developed with Tau-1 (20 µg of cell lysate per lane). Not shown in this figure, these treatments did not affect total tau levels significantly as determined by immunolabeling with Tau-1 after dephosphorylation. Only the GSK-3 inhibitors, lithium and VA increased Tau-1 staining. (E) GSK-3 inhibitors alsterpaullone and LiCl inhibit the GSK-3 enzyme activity in PC12 cells. PC12 cells were treated for 1.5 hours with medium (C), 10 µM butyrolactone I (Bu), 20 µM alsterpaullone (Alst) or 20 mM lithium chloride (LiCl). Cells were lysed and the GSK-3 enzyme activity determined. ^*P<0.001.