Fig. 2. Identification of TLR4 region essential for interaction with JIP3. (A) Schematic presentation of the mouse TLR4 mutants used in the assay. Black boxes represent transmembrane domains (TM). (B) C-terminal region of TLR4 is essential for the interaction with JIP3. HEK 293 cells were transiently transfected with expression plasmids of a Myc-tagged JIP3 (wild type or JNK-binding mutant) and an indicated Flag-tagged TLR4. At 48 h after transfection, JIP3 was precipitated with anti-Myc antibody and coprecipitation of TLR4 was detected with anti-Flag antibody. Lysates were also run as controls for the input. (C) Pro712 of TLR4 is not essential for JIP3 binding. HEK 293 cells were transiently transfected with expression plasmids of a Myc-tagged wild-type JIP3 construct and the TLR4 wild-type or Pro⁷¹²His point mutant. At 48 h after transfection, lysates were prepared and anti-Myc and control immunoprecipitates were analyzed for the coprecipitation of TLR4 with anti-Flag antibody.