TRIP promotes K48-linked ubiquitination and proteasomal degradation of TBK1. (A) Lysates from HEK293 cells transiently cotransfected with Flag-TRIP and Myc-TBK1, Myc-IRF3, or Myc-TRAF3 expression plasmids were subjected to immunoprecipitation with anti-Flag antibody followed by Western blot analysis with anti-Myc antibody. (B) Lysates from mouse peritoneal macrophages stimulated with LPS for indicated time periods were subjected to immunoprecipitation with anti-TBK1 or control IgG followed by Western blot analysis with anti-TRIP antibody. Proteins in whole-cell lysate were used as positive control (Input). (C) Schematic diagram of TRIP WT and mutant constructs. TRIP WT contains a RING domain, two coiled-coil domains, and a C-terminal CYLD interacting domain. In mutant CA, the cysteine residues at positions 7, 10, 25, 33, and 46 within the RING domain were substituted with alanine. In mutant ΔC51, the RING domain was simply deleted. (D) Lysates from HEK293 cells transiently cotransfected with Myc-TBK1, Flag-TRIP WT, or TRIP CA, and HA-Ub plasmids were subjected to immunoprecipitation with anti-Myc antibody followed by Western blot analysis with anti-HA antibody. (E) Lysates from HEK293 cells transiently cotransfected with Myc-TBK1, Flag-TRIP, or vector control, and HA-Ub (WT), HA-Ub (K48), or HA-Ub (K63) plasmids were subjected to immunoprecipitation with anti-Myc antibody followed by Western blot analysis with anti-HA antibody. (F) Western blot analysis of TBK1 expression in HEK293 cells cotransfected with HA-TBK1 and Flag-TRIP, Flag-TRIP ΔC51, or vector control. Similar results were obtained in three independent experiments in A, B, D, E, and F.