Fig. 1.

PIASy interacts with NF-κB p65 and represses TNF-induced NF-κB-mediated gene activation. (A) 293T cells were transiently transfected with Myc-p65 and Flag-PIASy, either alone or together as indicated. Whole-cell lysates were used for IP with anti-Flag, followed by Western blot with anti-Myc or anti-Flag. The same extracts were also subjected to Western blot using anti-Myc. (B) 293T cells and Raw264.7 cells were either untreated or treated with TNF (10 ng/ml) or LPS (100 ng/ml), respectively. Whole-cell lysates were used for IP with anti-PIASy, followed by Western blot using anti-p65. The same filter was reprobed with anti-PIASy. (C) Luciferase reporter assays. 293T cells were transiently transfected with a luciferase reporter (2× NF-κB), together with increasing amounts of Flag-PIASy. At 24 h after transfection, cells were either untreated or treated with TNF (10 ng/ml) for 6 h, followed by luciferase assays. The same extracts were analyzed by Western blot using anti-Flag.