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. 2013 Sep 19;8(9):e75372. doi: 10.1371/journal.pone.0075372

Figure 2. Substrate-induced Rsp5 auto-ubiquitination.

Figure 2

Western blot analysis of an in vitro Rsp5 ubiquitination reaction in the (A) presence (+CTD lanes) or (B) absence of substrate (-CTD lanes). Reactions (E1, E2, Rsp5, ATP, buffer, Ub,+/− CTD) were incubated from 15 to 30 minutes as indicated, and stopped by the addition of sample loading buffer. Both GST-Rsp5 and GST-CTD were detected with anti-GST antibody, and Rsp5 (unmodified and modified forms) detected on a separately run blot with anti-Rsp5 antibody. Asterisks in (B, upper panel) denote degradation products of Rsp5. These bands are not present in (A, upper panel), as they are obscured by the CTD band. Ubiquitinated and unmodified forms of Rsp5 and CTD are indicated.