Necdin inhibits PIAS1-dependent sumoylation of STAT1 and PML.

<p>(<b>A</b>, <b>B</b>) Inhibition of PIAS1-dependent STAT1 sumoylation by necdin. HEK293A cells were transfected with combinations of expression vectors for FLAG-tagged STAT1 (FLAG-STAT1, 3 µg) (<b>A</b>), HA-tagged SUMO1 (HA-SUMO1, 10 µg), Myc-tagged PIAS1 (Myc-PIAS1, 3 or 6 µg) and necdin (Necdin, 1 µg). Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody (FLAG) and immunoblotted (IB) with anti-HA antibody (HA). Expressed proteins were immunoblotted with antibodies to FLAG, Myc, necdin, HA, and γ-tubulin (Tub). Sumoylated STAT1 signals were quantified by densitometry and normalized with STAT1 (second panels) and PIAS1 levels (third panels)(<b>B</b>). (<b>C</b>, <b>D</b>) Inhibition of PIAS1-dependent PML sumoylation by necdin. HEK293A cells were transfected with combinations of expression vectors for FLAG-tagged PML (FLAG-PML, 3 µg), HA-tagged SUMO1 (HA-SUMO1, 4 µg), Myc-tagged PIAS1 (Myc-PIAS1, 3 or 6 µg) and necdin (Necdin, 1 µg). PML sumoylation was analyzed and quantified as above. Values (<b>B</b>, <b>D</b>) represent the mean ± SD (<i>n</i> = 3). **<i>p</i><0.01.