FIGURE 4.

D96N fails to interact with MyD88. HEK293T cells were plated to half confluence in six-well plates. After attachment, cells were transiently transfected with either TLR2-YFP (A), TLR4-CFP (B), or MyD88-CFP (C) and FLAG-tagged Mal WT or Mal D96N. Two days after transfection, supernatants were removed, and cells were lysed. Anti-GFP polyclonal antibody was used for immunoprecipitation (IP). Immunoprecipitates were loaded on a denaturing gel, transferred to nitrocellulose membrane, and visualized with a monoclonal anti-FLAG-horseradish peroxidase antibody. The immunoprecipitation of TLR2-YFP, TLR4-CFP, and MyD88-CFP was confirmed by immunoblotting the membrane with anti-GFP antibody. The same antibodies were used to check the level of expression of the proteins in whole cell lysates. IB, immunoblot.