Effect of MED on the ubiquitination of TRAF6 <i>in vivo</i> and <i>in vitro</i>.

<p>A. HEK 293T cells were transfected with Myc-TRAF6 and HA-Ub expression plasmids and incubated overnight, then cells were treated with various doses of MED for 4 h. Cell lysates were immunoprecipitated and analyzed by Western blotting. B. MED inhibited the ubiquitination of TRAF6 <i>in vitro</i> ubiquitination assay. HEK 293T cells were transfected with Myc-TRAF6 expression plasmids and incubated overnight, and then cell lysates were immunoprecipitated by TRAF6 antibodies for 3 h. Then the beads were washed three times using IP buffer and twice using TBS buffer. The beads were pre-treated with various doses of MED for 30 min on ice and then subjected to <i>in vitro</i> ubiquitination assay.