Ubiquitination of Stat5A in a cell-free system and identification of Ubc5 as the E2 for Stat5A ubiquitination. (A) Ubiquitination of Stat5A by Ubc5 and ¹²⁵I-ubiquitin in a cell-free system. Active Stat5 proteins were pulled down from 32D (EpoR wt) cell nuclear extracts using biotin-labeled double-stranded Stat5-binding oligodeoxynucleotides and avidin-conjugated Sepharose beads. The beads were incubated with human E1, an E2 as indicated, and ¹²⁵I-ubiquitin. Subsequently, the reaction products were subjected to SDS-PAGE, followed by autoradiography. (B) Ubiquitination of Stat5A by Ubc5 and nonradioactive ubiquitin in a cell-free system. The pulled down Stat5 proteins from panel A were incubated with human E1, an E2 as indicated, and nonradioactive ubiquitin. Subsequently, the reaction products were subjected to SDS-PAGE, followed by Western blot analyses with antiubiquitin (α-Ub) or anti-Stat5A (α-Stat5A) antibodies.