PIASy is an E3 SUMO ligase for DRIL1. (A) In vitro sumoylation assay performed with 35 S-labeled in vitro -translated wt DRIL1, in vitro -translated PIAS1, PIAS3, PIASy, PIASxα, PIASxβ or recombinant RanBP2 and incubated in a sumoylation mix containing purified E1, E2, ATP and SUMO1. 10% of the in vitro -translated PIAS1 (P1), PIAS3 (P3), PIASy (Py), PIASxα (Pxα), PIASxβ (Pxβ) were 35 S-labeled in vitro -translated for input control (inset). (-) for empty vector. (B) 293T cells co-transfected with wt DRIL1, T7-PIAS1, T7-PIASy, SUMO1 or SUMO2 constructs. Lysates were Western blotted using antibodies against DRIL1, T7 and β-actin as loading control. (-) for empty vector.