Fig. 3.

Length distribution of substrate-attached ubiquitin chains in yeast-soluble lysate. a Ub-ProT analysis of cellular ubiquitylated proteins. Exponentially growing cells were treated with or without 100 μM MG132 for 4 h. Ubiquitylated proteins in lysates were captured by TR-TUBE and subjected to trypsinization. b Composition of ubiquitin linkages in lysate, TR-TUBE-captured proteins, and Ub-ProT sample. For MG132-treated wild-type cells (n = 5) and sample pulled down (PD) with TR-TUBE (n = 4), the gel region above 49 kDa was subjected to Ub-AQUA/MS analysis (Supplementary Data 2). For Ub-ProT samples (n = 3), sum of Ub linkages quantified in d was represented (Supplementary Data 3). c Estimation of chain length of Ub-ProT samples. Gel mobility of free K48- and K63-linked chains (left; see Fig. S3 in detail). Anti-ubiquitin blot (middle) and protein staining (right) of the Ub-ProT sample. Here the amount of material analyzed was 5- or 15-fold higher than that in a, for the anti-Ub blot and protein staining, respectively. Gel regions subjected to MS quantitation are indicated by red letters (a–l). The position of the ubiquitin monomer (Ub₁) was defined as the gel fraction “a.” Asterisks denote TR-TUBE. d Length distributions of total ubiquitin and five major linkages at steady state or following MG132 treatment. Gel fractions in c were analyzed by quantitative mass spectrometry (mean ± s.e.m.; n = 3 biological replicates; Supplementary Data 3). Relative positions of K48- and K63-linked chains are labeled in blue and red, respectively