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Siah2 directly ubiquitylates TIN2 in vitro. Reaction mixtures containing the indicated combinations of E1, E2 (UbcH5), E3 (Sumo-Siah2), GST-TIN2, and HA-ubiquitin were incubated, fractionated on SDS-PAGE, and transferred to nitrocellulose. Proteins were visualized by staining with Amido black (right panel), and ubiquitylated proteins were detected by probing with anti-HA antibody (left panel).
