Figure 5. Effect of LRRK2-mediated phosphorylation on the ability of tau to bind to tubulin.

(A) Recombinant tau (2 µg) was incubated with GST-LRRK2 (50 ng) and 3 µCi of [γ-³²P]ATP in the presence of porcine tubulin (1 µg) at 30°C for 120 min and then a GST pull-down assay was performed as described in Materials and Methods. ³²P-Labeled proteins in the precipitates were detected by autoradiography following SDS-PAGE (upper panel). Total tau in the same sample was detected by Western blotting analysis using anti-tau antibody (lower panel). (B) V5-LRRK2 was immunoprecipitated with anti-V5-agarose beads from a WT4-D33 cell lysate. Precipitated proteins were analyzed by Western blotting using antibodies against V5-tag, phospho-tau (Thr181), non-phospho-epitopes of tau, and tubulin.