Fig. 2.

Scc2 dephosphorylation promotes amino-terminal cleavage. (A) Scc2-FLAG, immunopurified from asynchronously growing (1891-32C) cells, was incubated with WCE of untagged (1891-36D) cells staged at specific cell cycle intervals (indicated by min postαF release) or with pooled and boiled WCE. Following extensive washing, Scc2-FLAG species were analyzed via immunoblot (Upper). The ratios of slower:faster migrating forms of Scc2 were determined by using the immunoblot and plotted (Lower). (B) WCE of asynchronously growing Scc2-FLAG (1891-32C) cells was treated with lambda phosphatase or mock treated and analyzed by immunoblot using FLAG [Left (Upper and Lower are long and short exposures, respectively)] or a rabbit anti-Scc2 against the Scc2 amino terminus (Upper Right). Lower Right is a reprobing of the third image with FLAG. Carets, arrows, and asterisks indicate phosphorylated, full-length, and cleaved species of Scc2, respectively. (C) Scc2-FLAG immunopurified from WCE of asynchronously growing Scc2-FLAG cells (1891-32C) was treated with λ phosphatase before, or after, its isolation, and Scc2 species distributions were then determined by FLAG immunoblot.