Immunoblot analysis of whole-cell trichloroacetic acid lysates of indicated strains with specific Pex5p antibodies. (A) Mutant pex5Δ cells were transformed with plasmids expressing Pex5p_{C6K/K18R/K24R}. The ubiquitination pattern of the Pex5p-variant expressed in mutant pex5Δ cells is different from the Pex5p-modification pattern of pex1Δ cells, but resembles the pattern for Pex5p of a pex4Δ strain, although with shifted intensities. (B) Ubiquitination pattern of indicated Pex5p variants expressed in mutant pex5Δ and in double-mutant pex5Δpex4Δ cells. The pex4Δ-ubiquitination pattern is observed for Pex5p variants when Pex4p is absent, but is more pronounced than in the wild-type. In the Pex5pC6K variants, the second Ub-Pex5p band from below shows a small shift to a lower molecular mass.