Figure 5. Evidence that Ser⁹¹⁰/Ser⁹³⁵ phosphorylation is not mediated by autophosphorylation.

Endogenous LRRK2 was immunoprecipitated from Swiss 3T3 cells treated with DMSO, 30 μM H-1152 or 10 μM sunitinib for 2 h to induce dephosphorylation of Ser⁹¹⁰ and Ser⁹³⁵. Immunoprecipitates were washed multiple times with lysis buffer containing 0.5 M NaCl to remove inhibitor and were then incubated in kinase buffer containing 20 μM Nictide in the presence or absence of magnesium ATP (Mg:ATP) for 30 min. Following incubation, immunoprecipitates were centrifuged at 6000 g for 0.5 min and the supernatant was spotted on to P81 paper for measurement of LRRK2 kinase activity. Sample buffer was added to the pelleted beads and LRRK2 Ser⁹¹⁰ and Ser⁹³⁵ phosphorylation was quantified following immunoblot analysis with the indicated antibodies. A membrane was also subjected to autoradiography to assess LRRK2 autophosphorylation (³²P). The minor effect that H-1152 had on LRRK2 kinase assay is not significant.