Figure 4.

Down-regulation of 4E-BP1 phosphorylation in oocytes results in defects in the MII spindle assembly. (A) Scheme of dominant negative mutant construct of 4E-BP1–4Ala used for in vitro transcription. (B) Scheme of experimental procedure to express 4E-BP1 RNA constructs in the oocyte. (C) Immunoblotting evaluation of expression of microinjected non-phosphorylable form (marked by arrowhead) of 4E-BP1 in the matured MII oocytes n = 2. GAPDH was used as a loading control. See Figure S5. (D) Confocal images of MII spindles of oocytes microinjected with 4E-BP1-Wt or dominant negative mutant 4E-BP1–4Ala, Tubulin (red) and DNA (blue). Scale bar = 10 µm. (E) Quantification of chromosome alignment in the metaphase plate, MII oocytes expressing 4E-BP1-Wt or 4E-BP1-Ala RNA. Data are presented as mean ± SD, Student's t-test, n ≥ 25.