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. 2010 Apr 12;107(17):7704–7709. doi: 10.1073/pnas.0908764107

Fig. 3.

Fig. 3.

K63-polyubiquitylated PCNA is not a target of proteasomal degradation. (A) Inhibition of the proteasome by the chemical inhibitor MG132 causes an accumulation of total ubiquitin conjugates. Exponential cultures of HISPOL30 pdr5 cells were treated with 50 μM MG132 for 2 h where indicated, and ubiquitylated species were detected in total extracts by Western blots with an anti-ubiquitin antibody. Detection of phosphoglycerate kinase served as loading control. (B) Mutants with attenuated proteasome activity accumulate total ubiquitin conjugates. Extracts were prepared from the indicated strains and probed as in (A). (C) Damage-induced ubiquitylation of PCNA is reduced upon chemical inhibition of the proteasome. HisPCNA was isolated by denaturing Ni-NTA pull-down from extracts of HISPOL30 pdr5 cells treated with 50 μM MG132 for 2 h and 0.02% MMS for 90 min where indicated, and Western blots were developed with anti-PCNA and anti-ubiquitin antibodies. (D) Damage-induced ubiquitylation of PCNA is reduced in mutants affecting proteasome activity. HisPCNA and its ubiquitylated forms were isolated from the indicated strains and detected as in (C). The high-molecular weight signals in (C) and (D) marked with an asterisk are due to nonspecific isolation of ubiquitin conjugates; they are neither PCNA-reactive nor damage-dependent.