Fig. 4.

Cdc48/p97 regulates ubiquitin chain length. a Ub-ProT analysis of UPS-related mutant cells. Soluble lysate from wild-type (WT), ubp6Δ, rad23Δ dsk2Δ (r23Δ d2Δ), or CDC48 mutant (cdc48-3) cells were subjected to Ub-ProT analysis. Gel regions subjected to MS quantitation are indicated by red letters (a–l). Free ubiquitin chains were used as markers. b Absolute amounts of ubiquitin linkages in lysate from MG132-treated and cdc48-3 cells. The gel region above 49 kDa, where ubiquitylated proteins were detected, was subjected to Ub-AQUA/MS analysis (mean ± s.e.m.; n = 5 biological replicates; Supplementary Data 5). c Length distributions of total ubiquitin and five major linkages in MG132-treated wild-type or cdc48-3 cells. Gel fractions in a were analyzed by quantitative mass spectrometry (mean ± s.e.m.; n = 3 biological replicates; Supplementary Data 6)