FIGURE 3.

A52, but not A52-F154A, enhances TRAF6 self-association and causes TRAF6-TAK1 association. A, HEK293-TLR4 cells were transfected with empty vector (EV), Myc-A52, or Myc-A52-F154A. Cells were stimulated with 100 ng/ml LPS for 30 min 48 h after transfection. Lysates were analyzed by either native gel electrophoresis or SDS-PAGE. Immunoblotting was performed using standard conditions. Immunoblots (IB) were subjected to densitometric analysis with levels of the TRAF6 oligomer and phospho-p38 normalized to total levels of TRAF6 monomer and p38, respectively. B and C, HEK293T cells were transfected for 24 h with 4 μg of A52, A52-F154A, IRAK2, or HA-ubiquitin (Ub) as indicated. Endogenous TRAF6 was immunoprecipitated from cell lysates, and levels of ubiquitinated TRAF6 were assessed by immunoblotting with anti-HA antibody. HC, heavy chain of anti-TRAF6 antibody. D, HEK293T cells were transfected with Myc-TRAF6, FLAG-TAK1, HA-A52, and HA-A52 F154A. After 48 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. E, HEK293-Ts were transfected with 50 ng of Myc-A52 or pCMV-Myc (EV) and 100 ng of either TAK1 DN, MKK3 DN, or MKK6 DN, along with the pFR luciferase reporter gene and CHOP-Gal4. Luciferase reporter gene activity was measured 24 h after transfection. The data are mean ± S.D. of triplicate samples and are representative of at least three separate experiments.