FIGURE 1.

Ste4 undergoes ubiquitination in response to pheromone stimulation. Wild-type or ste4Δ cells were transformed with either empty vector or a plasmid expressing His-8-tagged ubiquitin (His8-Ubi). Early log phase cells were treated with 3 μm pheromone for the indicated time (min). His-8-Ubi-conjugated proteins were purified from whole cell lysates under denaturing conditions. The level of Ste4 in the whole cell extracts (WCE) prior to purification was detected by immunoblotting (IB) with anti-Ste4 antibodies (top panel). The purified samples were analyzed by immunoblotting with anti-Ste4 antibodies (center panel) and anti-ubiquitin antibodies (bottom panel). p-Ste4, phosphorylated Ste4; Ubi-Ste4, ubiquitinated Ste4.