FIG. 7.

Overexpression of PIASy-stabilized YY1 in vivo. (A) Coexpression of PIASy increases the steady-state level of YY1 protein. In Cos-7 cells, plasmid expressing HA-YY1 was cotransfected with an empty vector or 75 and 150 ng of Flag-PIASy plasmid. The steady-state protein levels of HA-YY1, PIASy, and the cotransfected GFP proteins were immunoblotted with anti-HA (F-7), anti-Flag (D-8), and anti-GFP (B-2; Santa Cruz) antibodies, respectively. (B) Overexpressed PIASy increases the stability of YY1. In Cos-7 cells, plasmid expressing HA-YY1 was cotransfected with an empty vector or Flag-PIASy plasmid. Two days posttransfection, the cells were treated with 10 μg/ml cycloheximide to block protein synthesis and the samples were collected at the time points labeled at the top. After the starting HA-YY1 levels in the two groups were normalized, the HA-YY1 protein at each time point was analyzed by Western blotting using the anti-HA antibody F-7. β-Actin immunoblotted by an anti-β-actin antibody (MAB1510; Chemicon) was used as the loading control.