Figure 4. TRAF3 SUMOylation is Ubc9-dependent.

(A) Over-expression of “dominant-negative” Ubc9^C93A which is unable to catalyze formation of SUMO conjugates reduces endogenous TRAF3 SUMOylation. HEK293 cells were transfected with Ubc9^C93A and lysates were obtained using an IDO-containing lysis buffer. Anti-TRAF3 immunoprecipitates (I.P) were immunoblotted (I.B) with either anti-SUMO-1 or anti-SUMO-2/3 antibodies, as indicated. Lysates were also immunoblotted with anti-MYC tag antibody to confirm expression of Ubc9^C93A or anti-TRAF3 to detect the levels of immunoprecipitated endogenous TRAF3. CV; control vector. (B) Knock-down of Ubc9 diminishes TRAF3 SUMOylation. HEK 293 cells were transfected with Ubc9 siRNA or an unrelated siRNA targeting luciferase (Luc) prior to lysis. Anti-TRAF3 immunoprecipitates were then immunoblotted with either anti-SUMO-1 or anti-SUMO-2 antibodies, as indicated. Results are representative of at least 4 independent experiments for (A) and (B).