Fig. 6

Phosphorylated C-terminus of Sae2 interacts with Rad50. a Full-length phosphorylated recombinant MBP-tagged pSae2 was mock-treated or dephosphorylated with λ phosphatase upon binding to amylose resin and incubated with recombinant MRX complex. The eluates were visualized by silver staining. Prescission protease was added to all samples as a protein stabilizer and to cleave the MBP tag off pSae2. b The FLAG-tagged recombinant Rad50 protein was immobilized on anti-FLAG affinity resin and incubated with phosphorylated C-terminal domain of pSae2 (pSae2 ΔN169, residues 170–345), which had been either mock-treated or dephosphorylated with λ phosphatase. The bound proteins were eluted and detected by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. c The phosphorylated recombinant MBP-tagged C-terminal domain of pSae2 (residues 170–345) was bound to amylose resin, eluted, cleaved with prescission protease, and immobilized on NiNTA resin. The bound pSae2 ΔN169 was mock-treated or dephosphorylated with λ phosphatase and incubated with recombinant wild-type Rad50 or ATP binding-deficient Rad50 K40A. Proteins were eluted and visualized by Ponceau staining or western blotting. Avidin was added to elution buffer and shows equal loading. d Assay as in c. Phosphorylated C-terminal domain of pSae2 was incubated with wild-type Rad50 or Rad50 K81I (representative Rad50S) mutant