FIG. 3.

The SAM domains of Anks family proteins associate with the ubiquitinated EphA8 receptor. (A) GST pulldown assay of ubiquitinated EphA8 using AIDA-1 SAM domains. Transfections were performed as described for Fig. 2B, except that an expression vector for HA-tagged ubiquitin (1 μg) was used. The arrow indicates the position of nonubiquitinated EphA8. (B) GST pulldown assay of EphA8-Ub using AIDA-1 SAM domains. Note that expression of EphA8-Ub was lower than that of wild-type EphA8 (third panel). (C) HEK293T cells were transfected with the indicated expression constructs. Cell lysates were subjected to immunoprecipitation using an anti-Odin antibody, and immune complexes were analyzed by WB using an anti-EphA8 antibody (top panel). The blot was reprobed to determine the levels of endogenous Odin (second panel). WCL were directly probed by WB to determine the levels of EphA8 and EphA8-Ub (third panel). (D) The data in panel C were quantitated, and the amount of EphA8 in association with Odin (panel C, top panel) was normalized to total EphA8 (panel C, third panel). Data represents the means ± standard errors (SE) from three independent experiments. EphA8-Ub associated with Odin more strongly than wild-type EphA8 (*, P < 0.001 by analysis of variance [ANOVA]).