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. 2013 Jul 29;208(11):1830–1840. doi: 10.1093/infdis/jit380

Figure 2.

Figure 2.

Ixodes scapularis XIAP is an E3 ubiquitin ligase. A, I. scapularis (Is) XIAP domains [(BIR: 61–135 aa., blue); (RING: 255–289 aa., green)] compared to related proteins in humans (Hs), mice (Mm), and Drosophila (Dm). UBA, ubiquitin associated domain (red). B–C, Ubiquitination assays followed by Western blot. Polyubiquitination assays were performed in the presence of ubiquitin, an E1 ubiquitin-activating enzyme (Ube1), E2 ubiquitin-conjugating enzymes (UbcH), and the tick recombinant E3 ubiquitin ligase XIAP expressed in Escherichia coli. Ten different E2 enzymes were used in (B), and UbcH13 was used as the E2 in (C and D). Aliquots were resolved in 12% SDS-PAGE and probed with an UbPan ubiquitin. Experiments were repeated at least twice. D, Polyubiquitination assays were performed in the presence of XIAP expressed in E. coli tagged with GST. Aliquots were resolved in 12% SDS-PAGE and probed for GST (upper panel) and XIAP (lower panel). Abbreviations: GST, glutathione-S-transferase; RING, really interesting new gene; XIAP, x-linked inhibitor of apoptosis protein.