MED suppresses TRAF6 polyubiquitination but not IRAK1 phosphorylation and TRAF6 dimerization.

<p>A. Effect of MED on polyubiquitination of TRAF6 induced by LPS in RAW264.7 cells. Cells were treated with LPS (100 ng/ml) together with various amounts of MED for 10 min. The cell lysates were immunoprecipitated by TRAF6 antibodies or isotype IgG, and then TRAF6 polyubiquitination was determined by Western blotting. B. Effect of MED on activation of IRAK1 induced by LPS in RAW264.7 cells. Cells were treated by LPS (100 ng/ml) together with MED (10 μM) for different times. The non-phosphorylated and phosphorylated IRAK1s (active form) were detected by Western blotting. ** p<i><</i>0.01. C. Effect of MED on dimerization of TRAF6 in HEK 293T cells overexpressing TRAF6. The cells were transfected with Myc-TRAF6 expression plasmids and incubated overnight, then the cells were treated with various doses of MED for 4 h. Cell lysates were analyzed by native gel and denature gel.