Fig. 4. Rsp5/ByS forms K63-linked polyubiquitin chains. (A) K63-linked polyubiquitin chains formed by Rsp5ΔWW and Tagless-Ub–MES were visualized by western-blotting with K63-ubiquitin linkage specific antibodies. A mixture of Rsp5ΔWW (1.8 μM) and each Ub–MES mutant was incubated for indicated times and analyzed by coomassie staining and western-blotting. MALDI-TOF analysis was performed for the higher MW bands produced in the reaction with Rsp5ΔWW and (B) Tagless-Ub(wt)–MES (lane 4), (C) Tagless-Ub(K48R)–MES (lane 7), and (D) Tagless-Ub(K63R)–MES (lane 10). The gel region of 100–250 kDa from lane 4, 7, and 10 was excised for MALDI-TOF analysis. Any peak corresponding to the calculated polyubiquitin linkage signal (Table S2–S4†) is marked in red. Autoubiquitinated Rsp5ΔWW is marked with *.
