Figure 3.

Both Res1 and Res2 remain associated with Cdc10 during a hydroxyurea block and release. (A and B) Exponentially growing (Exp) FLAGcdc10 cells (lanes 1 and 5) were treated with hydroxyurea (11 mM) for 4 h (lanes 2 and 6). Cells were released from the hydroxyurea block by washing out the hydroxyurea, and samples were taken after 0.5 h (lanes 3 and 7) and 1 h (lanes 4 and 8). (A) Samples were analyzed by Northern blotting for cdc18⁺ and his3⁺. (B) Whole-cell extracts prepared from cells treated as described above were subjected to immunoprecipitation with anti-FLAG antibody. Extracts (lanes 5–8) and immunoprecipitates (lanes 1–4) were subjected to Western analysis with anti-Cdc10 and anti-Res1 antibodies. (C) The level of Res2 and Cdc10 associated with Res1 does not change during an HU block and release. Exponentially growing (exp) wild-type cells (lane 1) were blocked with hydroxyurea (11 mM) for 4 h (lane 2) and then released from the block for 1 h (lane 3). Whole-cell extracts from these cells and from exponentially growing Δres1 cells (lane 4) were immunoprecipitated using monoclonal anti-Res1 antibody (Ayte et al., 1995) and probed by Western blotting with polyclonal anti-Res1, anti-Res2, and anti-Cdc10 antibodies. CO-I.P., coimmunoprecipitate; I.P., immunoprecipitate.