IKKα and IKKβ contribute to LRRK2 Ser935 phosphorylation following TLR activation.

<p>(<b>A</b>) Primary bone marrow derived macrophages were treated with either DMSO, 1 µM 5z-7-Oxozeanol (TAK1 inhibitor), 2 µM NG25 (TAK1 inhibitor), 2 µM MRT67307 (TBK1/IKKε inhibitor) or 10 µM B1605906 (IKKβ inhibitor) alone or in the indicated combinations for 1 h before stimulation with 100 ng/ml LPS for 1 h. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. (<b>B</b>) Bone marrow derived macrophages were generated from kinase inactive IKKα double Ser176A and Ser180A knock-in mice or littermate wild type controls. Macrophages were treated with either DMSO, 2 µM MRT67307 (TBK1/IKKε inhibitor) or 10 µM B1605906 (IKKβ inhibitor) as indicated for 1 h before stimulation with 100 ng/ml LPS for 1 h. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. (<b>C</b>) As in <b>A</b> except that BMDMs were treated with DMSO, 1 µM 5z-7-Oxozeanol (TAK1 inhibitor), 2 µM MRT67307 (TBK1/IKKε inhibitor) alone or in combination for 1 h before stimulation with 1 µg/ml Pam₃CSK4 or 200 µg/ml zymosan. (<b>D</b>) Ha tagged wild type and kinase inactive variants of IKKα IKKβ IKKε and TBK1 were expressed by transfection into HEK293 cells then immunoprecipitated by Ha-agarose. Kinases were used to phosphorylate bacterially expressed substrates GST-LRRK2 (882-1300), GST-IκBα (2-54) and GST-IRF3 (1-427) at 30°C for 30 min before reaction termination with sample buffer. 10% of the in vitro phosphorylation reaction was used for immunoblot analysis with the indicated antibodies. <b>E</b>) Baculovirus expressed TAK1 and IKKε were used to phosphorylate bacterially expressed substrates GST-LRRK2 (882-1300), GST-MKK6 (2-334) and GST-IRF3 (1-427) at 30°C for 30 min before reaction termination with sample buffer. 10% of the in vitro phosphorylation reaction was used for immunoblot analysis with the indicated antibodies or for autoradiograph.