Figure 3. Identification of phosphorylation site for LRRK2 in tau.

(A) ³²P-labeled phosphoamino acids of tau fully phosphorylated by GST-LRRK2 in the presence of tubulin were analyzed by two-dimensional thin-layer cellulose (TLC) electrophoresis, as described in Materials and Methods. (B) ³²P-labeled tau was separated by SDS-PAGE and digested with trypsin in the gel. The resulting polypeptides were separated by TLC electrophoresis (first dimension) and chromatography (second dimension). The phosphopeptides were loaded at the position indicated as the origin. (C) Tau was incubated with GST-LRRK2 and 100 µM non-radiolabeled ATP in the presence or absence of tubulin at 30°C for 180 min. The samples were then analyzed by Western blotting using antibodies specific to the phosphorylated Thr residues of tau.