FIG. 3.

Effect of overexpression of wild-type or mutant eIF2B on GSK-3β phosphorylation of tau. A total of 5 × 10⁶ PC12 cells were plated into 100-mm plates and transfected the following day with 9 μg of DNA using 100 μl of Lipofectamine Reagent/plate. Where indicated, cells were transfected with 1.2 μg of tau expression plasmid, 6 μg of GSK-3β expression plasmid, and 1.2 μg of expression constructs for wild-type or S535A mutant eIF2B, plus pcDNA3 to a total of 9 μg of DNA. One day after transfection, cells were lysed and 100 μg of proteins was analyzed by immunoblotting with a polyclonal anti-tau (pS³⁹⁶) phosphospecific antibody (0.4 μg/ml; BioSource) or with a monoclonal anti-tau antibody (1 μg/ml; BioSource).