Figure 5(A) Igo1 phosphorylation (P-Igo1) was followed by anti-MYC IB of protein extracts from WT, ppk18Η, and igo1-S64A strains, grown in rich conditions (R) or starved for 45 minutes (S). Igo1 migration was analyzed by electrophoresis of both a 12% SDS-polyacrylamide gel containing the Phos-tag™ molecule (right panel) and a standard 10% SDS-polyacrylamide gel (left panel). Arrowheads indicate the various phosphorylated isoforms of Igo1-MYC in each strain and condition. Shown is an IB representative of 2 independent experiments.(B,C) Expression of ste11+ (C) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, pab1Η, gcn5Η ppk18Η, pab1Η, pab1Η ppk18Η, igo1Η, igo1-S64A, gcn5Η igo1-S64A and pab1Η igo1-S64A. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).(B,C) Expression of mei2+ (D) using igo1 of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, ppk18Η, gcn5Η ppk18Η, pab1Η, pab1Η ppk18Η, igo1Η, igo1-S64A, gcn5Η igo1-S64A and pab1Η igo1-S64A. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).(D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT, S. pombeΗ, and igo1Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-MYC IB is shown as a control for loading. (D,E) The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. Shown below each blot are average measurements of 3 independent experiments (n = 3). A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. The star (*) symbol labels an unspecific band detected by the FLAG antibody in S. pombe.(E) P-Taf12 was followed in WT, igoS64A, pab1Η, and pab1Η igoS64A strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-Rpb1 IB is shown as a control for loading. (D,E) The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. Shown below each blot are average measurements of 3 independent experiments (n = 3). A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. The star (*) symbol labels an unspecific band detected by the S. pombe antibody in S. pombe.