FIGURE 1.
UFM1 activation and loading activity of UBA5. 0.5 μm UBA5, 1 μm UFC1, and 1 μm UFM1 were incubated in a 10-μl reaction buffer containing 50 mm Tris-HCl, pH 7, 5 mm MgCl2, in the presence and absence of 1 μm ATP or 5 mm dithiothreitol at room temperature for 90 min. Products were resolved by nonreducing 10–20% gradient SDS-PAGE and immunoblotted with anti-UFM1 rabbit polyclonal primary (Boston Biochem) and anti-rabbit secondary antibodies (Thermo Scientific) followed by enhanced chemiluminescent detection (GE Healthcare). *, 5 mm dithiothreitol added to assay mix following initial reaction incubation. x, 10 μm UFC1 or UFM1. Full-length (Uba5l) and truncated (Uba5s) reaction products are indicated. C, C-terminal, N, N-terminal.