Fig. 3

CDC25B distribution during oocyte maturation and interactions with YWHA proteins demonstrated by proximity ligation assays in oocytes and eggs. a Paired confocal images of equatorial sections through oocytes (left: immunofluorescence; right: brightfield) with rabbit anti-CDC25B. CDC25B is present thorught the oocyte and accumulates in the region of the nucleus across a two-hour time course during in vitro maturation of mouse oocytes. Control oocytes processed simultaneously in absence of the primary antibody showed minimal background fluorescence. These images are representative of two experiments that examined 6–12 cells at each time point. b Representative images of oocytes and eggs showing proximity ligation reaction spots that indicate interaction of YWHA protein isoforms with CDC25B at the molecular level. Each reaction spot represents a protein-protien interacton between a CDC25B and a YWHA protein (see Methods for details). Except for the lower left images, the upper panel is an oocyte and the lower panel is an egg, each imaged with confocal fluorescence microscopy. The equatorial scan is a single scan and the compressed Z is obtained from combining consecutive cross-sectional images throughout each cell taken at 3 μm intervals. The corresponding brightfield image is included. The lower two left panels are representative of the experiment using PLA with different primary antibodies against CDC25B directed at different sites on the protien. Binding to the same protein is indicated by PLA spots (rabbit anti-CDC25B and goat anti-CDC25B; rabbit anti-pSer149 CDC25B and goat anti-CDC25B). No reaction spots are observed in the control (oocytes and eggs processed in absence of the primary antibodies). The images shown are representative of two experiments (oocytes and eggs from four animals) with similar results for a total of 7–10 oocytes and a total of 6–11 eggs for each condition and controls. Scale bars for all images represent 10 μm