Fig 2. Tup1 and Cyc8 are sumoylated during hyperosmotic stress.

(A) Scheme of the MS strategy to identify proteins sumoylated during hyperosmotic stress. (B) Total peptide counts identified for Tup1 and Cyc8 at 0 and 15 minutes of hyperosmotic stress. Total peptide counts for Smt3 are included to demonstrate equivalent levels of SUMO in the samples. (C) Total peptide counts identified for proteins where the significance of the changes between 0 and 15 minutes of hyperosmotic stress was p≤0.05. Gray areas represent ≥3 fold changes in the 15 minute samples compared with the 0 minute samples. Tup1 and Cyc8 are noted. (D) Cells expressing His₆-FLAG-Smt3 (HF-Smt3) and either a 3xHA epitope-tagged Tup1 (Tup1-3HA) or a 3xHSV epitope-tagged Cyc8 (Cyc8-3HSV) from their endogenous promoters were subject to hyperosmotic stress (1.2M sorbitol) over a 60-minute time course. Cell lysates (input) and purified sumoylated proteins (SUMO pulldown) were subject to western analyses using anti-HA, anti-HSV, or anti-Pgk1 antibodies to detect Tup1, Cyc8, or Pgk1 respectively. (E) Similar experiment as in (D) except cells were subject to hyperosmotic stress (1.2M sorbitol), heat shock (42°C), or high ethanol (10% v/v) for 0, 15, and 60 minutes.