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. 2008 Mar 31;28(12):4196–4203. doi: 10.1128/MCB.00169-08

FIG. 3.

FIG. 3.

Progesterone regulates the association of INCENP with Aurora B. (A) Ectopic INCENP activates Aurora B only after progesterone treatment. Oocytes were injected with mRNA encoding Myc-INCENP-IN and were incubated overnight; then some were treated with progesterone to induce maturation. Extracts from immature (IM) or progesterone-treated (meta-I) oocytes were immunoblotted with anti-Myc antibodies. The same extracts were analyzed for total Aurora B kinase activity in immunoprecipitates (IP), as indicated. Similar results were obtained in several independent experiments. (B) INCENP promotes T-loop phosphorylation of Aurora B. Oocytes were injected with mRNAs encoding both Myc-INCENP-IN and Flag-Aurora B, and some were then treated with progesterone. The expression of INCENP and Aurora B was monitored with antibodies to Myc or Flag, as indicated, and the phosphorylation of T248 (pT248) was measured with a phosphospecific antibody as described in Materials and Methods. (C) Association of INCENP and Aurora B during maturation. Oocytes were injected with mRNAs encoding Myc-INCENP and were incubated overnight; then some were treated with progesterone. Extracts from IM, meta-I (GVBD), and meta-II oocytes before (−) and after (+) Aurora B immunodepletion were immunoblotted with anti-INCENP and anti-Aurora B antibodies, as indicated. Mock depletion was performed with Dyna beads coupled to preimmune serum. (D) Aurora B T-loop dephosphorylation is blocked by INCENP. Xenopus CSF-arrested meta-II egg extracts were immunoblotted with anti-Aurora B or anti-phospho-T248 Aurora B antibodies at the indicated times after the addition of recombinant Aurora B alone or of wild-type or KN Aurora B bound to INCENP. (E) The association of Aurora B with PP1 is reduced during oocyte maturation. Extracts from IM and progesterone-treated meta-I (GVBD) oocytes were incubated with control rabbit immunoglobulin G or anti-PP1 antibodies bound to Sepharose beads; the beads were then washed and eluted with SDS sample buffer, and the eluates were analyzed by SDS-PAGE and immunoblotting with antibodies to PP1γ1 and Aurora B, as indicated.