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. 2015 Nov 9;211(3):653–668. doi: 10.1083/jcb.201504073

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© 2015 Ullal et al.

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Figure 5. — Pom1 inhibits Cdc15 binding with Fic1 and Pxl1. (A) Cells of indicated genotypes treated with 1 µM 3MB-PP1 (top) or no drug (bottom) for 2 h at 25°C, and stained with calcofluor. Bar is 5 µm. (B) Quantification of tip septa in septated cells as in A (n > 200). (C) Pom1 phosphorylation inhibits Cdc15-Fic1 interaction. Recombinant Fic1-His was immobilized on nickel beads and incubated with recombinant MBP or MBP-Cdc15C, prephosphorylated or not by recombinant GST-Pom1. Beads were washed and bound proteins were run on SDS-PAGE and detected by Coomassie-staining and Western blotting using anti-MBP antibodies. One fifth of each MBP protein input is shown as control. (D) Recombinant Fic1-His was immobilized on nickel beads and incubated with recombinant MBP, MBP-Cdc15C^19A, or MBP-Cdc15C^19D. Beads were washed and bound proteins were run on SDS-PAGE and detected by Coomassie staining and Western blotting using anti-MBP antibodies. One fourth of each MBP fragment input is shown as control. (E) Pom1 phosphorylation inhibits Cdc15-Pxl1 interaction. Recombinant GST-Pxl1 was immobilized on glutathione beads and incubated with recombinant MBP, MBP-Cdc15C, prephosphorylated or not by recombinant purified GST-Pom1 or GST-Pom1^kd, MBP-Cdc15C^19A, or MBP-Cdc15C^19D. Beads were washed and bound proteins were run on SDS-PAGE and detected by Coomassie staining and Western blotting using anti-MBP antibodies. One fifth of each MBP protein input is shown as a control.