Figure 2. MIB1 promotes RYK ubiquitination. (A) Western blot analyzing metal-affinity purification of His-tagged ubiquitin under denaturing conditions. The blot shows that that MIB1 can promote the ubiquitination of the N-terminal glue-tagged RYK, RYKICD, and membrane-tethered MYR-RYKICD but not RYK lacking the ICD (RYKΔICD). Mutant MIB1, C997S, and truncated MIB1, ΔRN3, have little or no catalytic activity with respect to RYK. (B) Western blot analyzing streptavidin purification of glue-RYKICD. The blot shows the conjugation of endogenous ubiquitin promoted by MIB1 and by nonphosphorylatable MIB1-S807A, but not by MIB1-C997S. (C) Western blot analyzing metal affinity purification of His-tagged ubiquitin under denaturing conditions showing that MIB1 can promote the ubiquitination of C-terminal myc-tagged mouse RYK, RYKICD, and noncleavable RYK-RC. Blots represent n > 3. The asterisk indicates a nonspecific band.
Figure 2.

MIB1 promotes RYK ubiquitination. (A) Western blot analyzing metal-affinity purification of His-tagged ubiquitin under denaturing conditions. The blot shows that that MIB1 can promote the ubiquitination of the N-terminal glue-tagged RYK, RYKICD, and membrane-tethered MYR-RYKICD but not RYK lacking the ICD (RYKΔICD). Mutant MIB1, C997S, and truncated MIB1, ΔRN3, have little or no catalytic activity with respect to RYK. (B) Western blot analyzing streptavidin purification of glue-RYKICD. The blot shows the conjugation of endogenous ubiquitin promoted by MIB1 and by nonphosphorylatable MIB1-S807A, but not by MIB1-C997S. (C) Western blot analyzing metal affinity purification of His-tagged ubiquitin under denaturing conditions showing that MIB1 can promote the ubiquitination of C-terminal myc-tagged mouse RYK, RYKICD, and noncleavable RYK-RC. Blots represent n > 3. The asterisk indicates a nonspecific band.

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