Figure 5. TANK controls TRAF6 ubiquitination in response to TLR7 stimulation in macrophages.
(a) Peritoneal macrophages from wild-type (WT) and Tank−/− mice were stimulated with 10μM R-848 for the indicated periods. Cell lysates were prepared and immunoprecipitated with anti-IRAK1. The kinase activities in the immunoprecipitates were measured using an in vitro kinase assay. (b) Macrophages from wild-type and Tank−/− mice were stimulated with 10 μM R-848 for the indicated periods. Whole cell lysates were subjected to immunoblot analysis with anti-IRAK1. Immunoblots ofβ-tubulin are shown as a loading control. (c) Cell lysates of macrophages treated with R-848 for the indicated periods were immunoprecipitated with anti-TRAF6, followed by immunoblot analysis with anti-Ub. Immunoblots of TRAF6 are shown as a loading control. Data of two independent experiments are shown. (d) HEK293 cells were cotransfected with Flag-TRAF6 and Myc-TANK. Cell lysates were immunoprecipitated with anti-Flag, followed by immunoblot analysis with anti-Ub. Immunoblots of β-tubulin are shown as a loading control. The data shown are representative of three independent experiments.