Fig 5. Phosphatase treatment of Ndt80 with and without Mek1-as or Ime2ΔC241-as kinase activity.

For all of the experiments in this figure, diploids were transferred to Spo medium for five hours, 1 μM ED was added (indicated by arrowheads) and the culture split in two. Either 1 μM 1-NA-PP1 (mek1-as) or 50 μM 3-MB-PP1 (IME2ΔC241-as) was added to one of the cultures and samples assayed at various timepoints. (A) Meiotic progression in dmc1Δ mek1-as NDT80-IN (NH2437::pEP105²::pBG4²) without (-I) and with (+I) 1-NA-PP1. Values represent the average of two independent experiments with error bars indicating the range. (B) Extracts generated from cells at the 0, 5 and 6 hr (- and + I) timepoints from one of the timecourses shown in (A) probed with antibodies recognizing the indicated proteins. (C) Phosphatase treatment of the 6 hr (- and +I) extracts. AP = alkaline phosphatase. p-Ndt80 indicates phosphorylated Ndt80; pr-Ndt80 = phosphatase resistant form of Ndt80; Ndt80 = unphosphorylated Ndt80. AP was preincubated with phosphatase inhibitors (AP Inh.) for 30 minutes prior to addition to extracts. Numbers indicate where the prestained molecular weight markers (in kD) ran on the gel. (D) Meiotic progression in DMC1 IME2ΔC241-as NDT80-IN and dmc1Δ IME2ΔC241-as NDT80-IN (yJL92 and NH2451, respectively) without (-I) and with (+I) 3-MB-PP1 as in Panel A. (E) Extracts from the indicated timepoints from one of the dmc1Δ IME2ΔC241-as NDT80-IN timecourses shown in D probed with antibodies recognizing the indicated proteins. (F) Phosphatase treatment of the 7 hour extracts shown in (E).