Fig. 7. Mos targets Myt1. (A) Mos associates with Myt1 in vivo: stage VI oocytes were injected with mRNA encoding GST protein, GST-Mos wild type (WT) or GST-Mos kinase dead (KD) protein. In some cases, as indicated, oocytes were incubated in U0126 during the whole experiment. Twelve hours after microinjection, oocytes were treated (or not) with progesterone. Four hours later, groups of 40 oocytes were collected and submitted to a GST pull-down assay. The elution of the beads was analyzed by immunoblot with the indicated antibodies. The equivalent of one immature oocyte (stage VI) and one mature oocyte (control M) are shown as control. (B) In vitro phosphorylation of Myt1. mRNAs of GST-Mos WT or GST-Mos KD (as a negative control) were injected into stage VI oocytes. Fourteen hours later, extracts of 30 oocytes of each type were prepared. Myt1 translated in reticulocyte extract (without radiolabeled amino acids) was added (20 µl) to the GST-pull down performed from injected oocytes. Myt1 phosphorylation was tested in presence of ³²PATP, MgCl₂ and microcystin. After several washes of the beads, the samples were loaded on a 8% acrylamide gel and analyzed by autoradiogram and western blotting using anti-Myt1 antibodies. Lanes 1-4: extracts (equivalent of one oocyte) of stage VI oocytes (lane 1), metaphase II oocytes (lane 2), GST-Mos KD-injected oocytes (lane 3), and GST-Mos WT-injected oocytes (lane 4). Lanes 5,6: GST pull-down on oocytes injected with GST-Mos KD (lane 5) or Mos WT (lane 6) after the phosphorylation reaction. Lanes 7-9: Myt1 translation in vitro without radiolabeled amino acids (lane 7; 4 µl), with ³⁵S Met (lane 8; 2 µl) or without Myt1 plasmid (lane 9; 2 µl negative control). Lane 8 is a control for Myt1 translation and for the size of non phosphorylated Myt1.