FIG. 4.
Modification of ARIP3 and other PIAS proteins by SUMO-1 in mammalian cells and in vitro. (A) COS-1 cells grown on 6-cm-diameter dishes were transfected with 1 μg of pFLAG-ARIP3, pFLAG-Miz1, pFLAG-PIAS1, or pFLAG-PIAS3 and 1.5 μg of empty pSG5 vector or pSG5-His-SUMO-1. Forty-eight hours after transfection, the cells were lysed in RIPA-1 buffer containing 10 mM NEM, and the cell lysates were immunoblotted with the M2 FLAG antibody. (B) Sumoylation of PIAS proteins in vitro. In vitro-translated, 35S-labeled PIAS proteins were incubated with purified GST-SUMOGG-1 in the presence (+) or absence (−) of GST-SAE1 plus GST-SAE2 and GST-Ubc9 as indicated. Reactions were stopped by adding SDS-PAGE sample buffer, and the samples were resolved by SDS-PAGE and subjected to fluorography. (C) Sumoylation of purified GST-ARIP3, GST-ARIP3Δ347-418, and GST-ARIP3Δ467-487 (1 μg each) under conditions described for panel B. The reaction mixtures were immunoblotted with an anti-ARIP3 antibody.