Endogenous RelA SUMOylation by PIAS3 is induced by NF-κB activation.

<p>A) HEK 293T were transfected with his-SUMO3 plus PIAS3 or GFP control plasmid as indicated. NF-κB activation was achieved by either co-transfection of constitutive IKK2 or TNFα treatment (20 ng/ml for 4 hours). SUMOylated RelA was measured by nickle pull down followed by immunoblotting with anti-RelA antibody. B) NF-κB repression by PIAS3. The NF-κB activity of cells described in panel A was measured by NF-κB luciferase assay. C) The time course of RelA SUMOylation in response to NF-κB activiation. HEK 293T cells were transfected with his-SUMO3 plus PIAS3 or control GFP plasmid as indicated. The cell lysates were collected for <i>in vivo</i> SUMOylation assay at indicated time points after TNFα treatment (20 ng/ml). SUMOylated RelA was detected by nickel pull down followed by immunoblotting with anti-RelA antibody.