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. 2012 Oct 3;7(10):e46398. doi: 10.1371/journal.pone.0046398

Figure 3. MultiDsks can be used to characterise the kinetics of ubiquitylation of a specific protein species. A.

Figure 3

Exponentially growing yeast cells were either treated with 10 µg/ml of 4-NQO for one hour, or left untreated, as indicated. Equal amounts of extracts were incubated with MultiDsk resin. Dilute Input extract (1%) and washes from the beads were also loaded. Proteins were analysed by Western blotting using anti-Rpb1 antibody, 4H8. B. Exponentially growing yeast cells were either treated with 10 µg/ml of 4-NQO for one hour, or left untreated, as indicated. Equal amounts of the extracts were incubated with agarose beads loaded with GST alone, GST-Dsk2 protein, commercial TUBE1, or MultiDsk. Proteins were eluted via boiling in sample buffer and subjected to Western blot analysis using the anti-Rpb1 antibody, 4H8 (upper panel). Ponceau S staining (lower panel) shows relative amounts of affinity proteins used. C. Yeast cells were harvested at the indicated time after treatment with 4-NQO (10 µg/ml), incubated with MultiDsk resin, and isolated proteins were analysed by Western blotting using either 4H8, or an anti-Def1 antibody, as indicated.