Figure 4

Phosphorylation of tau in adult mouse brain slices treated with recombinant SET protein or Jcasp peptide for 5 h 30 min and its relationship with methylated PP2A. A) Proteins were extracted from mouse adult brain slices treated with recombinant SET protein or Jcasp peptide and were analyzed by western blotting (40 μg per lane). Representative western blots of tau hyperphosphorylation at Ser-202, Ser-404 and Ser-422 and total tau protein are shown. Brain slices not treated with any protein (Ctrl) and brain slices treated with mutated Jcasp (JA) were used as negative controls. Okadaic acid (OA) was used as positive control. PP2A was also analyzed. Actin was used as internal loading control. Jcasp or SET internalization induces hyperphosphorylation of the same tau isoforms. B) Quantification of hyperphosphorylation of Ser-202 (n = 8 independent experiments) and Ser-422 (n = 7 independent experiments) in brain slices treated with SET relative to the phosphorylation of tau at these residues in control cells (set to 100%). Results were normalized to total tau. C-D) Correlation between tau hyper-phosphorylation at Ser-202 (C) (n = 8 independent experiments) and Ser-422 (n = 7 independent experiments) (D) and methylated PP2A. Phosphorylation at Ser-202 is significantly negatively correlated with methylated PP2A in cells containing recombinant SET.