Figure 2. Evidence that LRRK2 kinase activity controls Ser⁹¹⁰ and Ser⁹³⁵ phosphorylation, as well as 14-3-3 binding.

(A and B) HEK-293 cells transiently expressing the indicated forms of FLAG–LRRK2 were treated with DMSO vehicle control or indicated concentrations of H-1152 or sunitinib for 90 min. Cells were lysed in lysis buffer supplemented with 0.5% NP-40 and 150 mM NaCl and subjected to anti-FLAG immunoprecipitation. Immunoprecipitates were resolved by SDS/PAGE (4–12% Novex gels) and subjected to immunoblotting with anti-FLAG (total LRRK2), anti-phospho-Ser⁹¹⁰ (αpS910), anti-phospho-Ser⁹³⁵ (αpS935) antibodies, as well as a 14-3-3 overlay assay, as indicated. Similar results were obtained in two separate experiments.