Fig. 5. The Cdc14 phosphatase dephosphorylates Lte1 in vivo and in vitro. (A) A strain expressing endogenous HA3-tagged Lte1 and carrying a GAL1-inducible CDC14 plasmid (SY138) was treated as described in Fig. 4A. At the indicated times, samples were withdrawn for protein analysis, Clb2-associated kinase assays using Histone H1 as substrate and FACS analysis. The arrow indicates position of unphosphorylated Lte1. (B) Similar analysis to that described in A was performed on a strain expressing Lte1HA3 and carrying a GAL1-inducible SIC1 plasmid (SY137). Multiple Lte1 forms are indicated with a bracket. An unspecific band seen with the anti-Sic1 antibody is indicated with an asterisk (C). Extracts from nocodazole-arrested cells expressing endogenous Lte1 HA3 tagged protein were subjected to immunoprecipitation using anti-HA antibody. The immunoprecipitates were left untreated (lane 1), incubated with 1 µg purified MBP-Cdc14 (lane 2), 1 µg MBP-Cdc14C283A (lane 3) or 1 µg MBP (lane 4).