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. 2014 Jun 24;289(31):21760–21769. doi: 10.1074/jbc.M114.550871

FIGURE 5.

FIGURE 5.

Requirement of the YPEL motif of Rfu1 for ubiquitin homeostasis. A, immunoblot analyses of wild-type, or Δrfu1 cells harboring a vector or a plasmid expressing Rfu1-GFP or Rfu1(Y190A,P191A)-GFP. Rfu1(Y190A,P191A)-GFP is indicated as Rfu1(AA)-GFP. Top, anti-ubiquitin immunoblot analysis. The position of size standard in kilodaltons is indicated on the left. Monomeric ubiquitin and ubiquitin chain positions are marked. Middle, phosphoglycerate kinase (PGK) immunoblot. Bottom, anti-GFP immunoblot. B, impaired association of Rfu1(Y190A,P191A)-GFP with endogenous Bro1 in vivo. Lysates of Δrfu1 cells harboring a vector or plasmids expressing Rfu1-GFP or Rfu1(Y190A,P191A)-GFP were immunoprecipitated (IP) with anti-GFP. The resulting immunocomplexes were analyzed by immunoblot using anti-GFP and anti-Bro1.