Mutant pex5Δ cells were transformed with plasmids expressing Pex5p or indicated variants. (A) Immunoblot analysis of equal amounts of whole-cell trichloroacetic acid lysates of indicated strains with Pex5p-specific antibodies. In contrast with the wild-type, pex5Δ expressing Pex5p_C6A shows up to three additional αPex5p reactive bands. The third and especially the second band are more pronounced in case of Pex5p_C6K and Pex5p_{C6K/K18R/K24R}. (B) The pex5Δ strain expressing Pex5p_{C6K/K18R/K24R} was additionally transformed with a plasmid either encoding ubiquitin (Ub) or myc-tagged ubiquitin (mycUb). The observed shift to a higher molecular mass upon mycUb expression proved that the additional αPex5p-reactive bands represent ubiquitinated Pex5p.