Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A -- Wu et al. 104 (42): 16564 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Wu et al. 10.1073/pnas.0707537104.

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SI Figure 5
SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9

SI Figure 5

Fig. 5. The Mos-induced mitotic arrest in cycling extracts is Ca²⁺-sensitive. Cycling extracts were incubated at room temperature for 45 min and then supplemented with MBP-Mos. Once arrested in mitosis, extracts were treated with buffer containing or lacking calcium. Samples were taken at the indicated times after calcium addition and immunoblotted for cyclin B2.

SI Figure 6

Fig. 6. Emi2 antisense morpholino oligonucleotides specifically repress the reaccumulation of Emi2 in cycling extracts. Cycling extracts were incubated at room temperature with [³⁵S]Met/Cys in the presence of control or Emi2 morpholino antisense oligonucleotides for 45 min (see ref. 11 for morpholino details). After incubation, the newly synthesized cyclin B2 (Left) or Emi2 (Right) was immunoprecipitated, washed, and detected by autoradiography.

SI Figure 7

Fig. 7. Mos/MAPK activity regulates the phosphorylation status of Emi2. (A) Radiolabeled IVT Emi2 was added to cycling extracts. After 45 min, MBP or MBP-Mos protein was added. Samples were withdrawn at indicated times for SDS/PAGE to detect ³⁵S-labeled Emi2 by autoradiography and pMAPK and pCdc2 by immunoblotting. (B) Flag-tagged, radiolabeled Emi2 was added into mitotic extracts. After 30 min, Emi2 was immunoprecipitated with anti-Flag antibodies, washed, and incubated +/- l-phosphatase for 30 min. ³⁵S-labeled Emi2 was detected by SDS/PAGE and autoradiography. (C) CSF extract was treated with DMSO or indicated doses of U0126 for 2.5 h. Emi2 protein and active MAPK were detected by immunoblotting, and Cdc2 kinase activity toward histone H1 was detected by using [g-³²P]ATP and autoradiography.

SI Figure 8

Fig. 8. A Cdc2-dependent mechanism of Emi2 destruction. (A) Indicated fragments of Emi2, progressively truncated at the C terminus, were in vitro translated in the presence of [³⁵S]methionine and incubated in CSF extract for the indicated amounts of time. Radiolabeled Emi2 was detected by SDS/PAGE and autoradiography. (B) Radiolabeled in vitro translated Emi2 (amino acids 1-279, WT, or the degron mutant, DS33AA) was added to CSF extracts. During room temperature incubation, samples were taken at the indicated times, and the levels of ³⁵S-labeled Emi2 (1-279) were examined by SDS/PAGE and autoradiography. (C) Emi2 (amino acids 1-279 WT, S213A, T239A, T252A, T267A, or 4AP) proteins were added to CSF extracts and processed as in B. (D) Radiolabeled in vitro translated Emi2 (WT or 4AP) proteins were added into CSF extracts supplemented with 100 nM cyclin B1. ³⁵S-labeled Emi2 remaining in the extract at each time point was examined by SDS/PAGE and autoradiography.

SI Figure 9

Fig. 9. The same region of Emi2 that confers stability in CSF extract interacts with PP2A. (A) GST or GST-SD (Emi2 residues 319-375) prebound to glutathione Sepharose was incubated in CSF extracts for 20 min. The beads were washed five times with PBS + 300 mM NaCl and 0.1% Triton, and levels of bound PP2A were examined by immunoblotting. (B) Radiolabeled IVT Emi2 (1-350 or 1-321) was added into interphase extracts pretreated with Mos protein. After 30 min incubation at room temperature, endogenous PP2A was immunoprecipitated and washed, and bound Emi2 was examined by SDS/PAGE and autoradiography.