Figure 5. Ser935 phosphorylation during TLR signaling does not require LRRK2 kinase activity.

(A) Primary bone marrow derived macrophages were treated with indicated concentrations of LRRK2-IN1 or CZC25146 or DMSO as control for 1 h. Cell lysates were prepared and subjected to immunoblot with the indicated antibodies. (B) Primary bone marrow derived macrophages were pre-treated with indicated concentrations of LRRK2-IN1 or CZC25146 or DMSO as control for 1 h before stimulation with 100 ng/ml LPS for 1h. Cell lysates were prepared and subjected to immunoblot with the indicated antibodies. (C) As in B except 1 µg/ml Pam₃CSK₄ was used. (D) RAW264.7 cells were treated with 100 ng/ml LPS or 1 µg/ml Pam₃CSK₄ for 1 h before cell lysis. Endogenous LRRK2 was immunoprecipitated from 3 mg RAW264.7 cell lysate with 3 µg rabbit monoclonal LRRK2 100–500 antibody. Kinase activity was measured using 20 µM Nictide with n = 4 in duplicate. Kinase activity is reported as pmol of ATP incorporated into Nictide per minute per mg of lystate immunoprecipitated from. A parallel set of immunoprecipitations was used to measure 14-3-3 binding by overlay assay. All results are representative of at least two independent experiments.