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. 2022 Mar 2;50(6):3276–3291. doi: 10.1093/nar/gkac135

Figure 3.

Figure 3.

Polyubiquitinated Cse4 is enriched in cdc48-3, ufd1-2, and npl4-1 mutants under normal physiological conditions. (A) Levels of Cse4 polyubiquitination, which are not specific to any cell cycle stage, are enhanced in cdc48-3, ufd1-2 and npl4-1 strains. Ub pull-down assay was performed using protein extracts from logarithmically growing cells, G1 phase cells synchronized with α-factor, S phase cells synchronized with hydroxyurea (HU), and M phase cells synchronized with nocodazole (Noc), in YPD at 25°C. The cell cycle arrest was verified by FACS analysis and cell morphology using propidium iodide-stained cells (Supplementary Figure S9). Input and ubiquitin pull down samples were analyzed using anti-HA (Cse4) and anti-Tub2 antibodies. Untagged Cse4 was used as a negative control. Asterisk shows nonmodified Cse4. (B) Levels of Cse4 polyubiquitination are enhanced by inactivation of Cdc48. Control (CDC48) and cdc48-aid strains were grown in YPD to logarithmic phase at 25°C, then treated with auxin at the indicated time point. For M phase arrested cells, nocodazole (Noc) was added to the media after 2 h auxin treatment. Ub pull-down assay was conducted as described in (A).