Figure 2: Bendless-XIAP molecular interactions.
From: Infection-derived lipids elicit an immune deficiency circuit in arthropods
(a) Protein differential expression on A. phagocytophilum infection of I. scapularis ticks based on iTRAQ proteomics data deposited on the Dryad repository database. (b) Structural docking model demonstrating the interaction between I. scapularis XIAP and the UbcH13 homologue, Bendless. (c,d) Native gel and ELISA analysis of a fixed amount of recombinant (0.2 μg) XIAP incubated with increasing amounts of recombinant Bendless. The analysis shown is one of two biological replicates. (e) XIAP-Bendless binding inhibition with a monoclonal antibody against the human homologue of Bendless (UbcH13). (d,e) The average of two technical replicates are plotted. (f) XIAP polyubiquitylation assay with recombinant Bendless (lane 1). Control conditions were performed in the absence of an E1 enzyme—Ube1 (lanes 2 and 6), XIAP (lanes 3, 5 and 6), wild-type ubiquitin (lanes 4, 11 and 12), Bendless (lanes 6, 7 and 9) and Uev1a (lanes 6, 8 and 9). Immunoblots were probed with antibodies specific for K63- (lanes 1–9) and K48- (lane 10) polyubiquitin chains or with a pan-ubiquitin antibody (lanes 11 and 12). GST was used as a negative control. The Western blot (WB) shown is one of two biological replicates. (g) Immunoprecipitation (IP) analysis followed by WB showing the interaction between XIAP and Bendless within HEK293 T cells transfected with the indicated vectors. Input indicates normalizing amounts. The WB shown is one of two biological replicates. (h) ELISA with protein extracts from unfed I. scapularis nymphs that were microinjected with either a scrambled control (scBendless) or siRNA specific for bendless (siBendless) and incubated with increasing amounts of recombinant XIAP. Points are the average of 10 biological replicates with two technical replicates each. See also Supplementary Figs 1–3 and 8–10.
