Fig 3. Tup1 and Cyc8 need to be in a complex together for hyperosmotic stress-dependent sumoylation.
(A) His6-FLAG-SMT3 wild-type, tup1Δ, and cyc8 cells were subjected to hyperosmotic stress (1.2M sorbitol) over a time course of 60 minutes. Changes in sumoylation patterns were examined by western analysis using an anti-FLAG antibody. (B-C) His6-FLAG-SMT3 promoters were subject to hyperosmotic stress (1.2M sorbitol) for 15 minutes. Cell lysates were generated and subject to metal affinity chromatography to purify sumoylated proteins. Cell lysates (input) and purified sumoylated proteins (SUMO pulldown) were subject to SDS-PAGE and western analyses using anti-HA (Tup1) or anti-HSV (Cyc8) antibodies. (B) Results for Tup1-3HA sumoylation when the Cyc8-interaction region was deleted in Tup1. (C) Results for Cyc8-3HSV sumoylation when the Tup1-interaction region was deleted in Cyc8.