Figure 2
Ribosome loading onto Mos, Ccnb1, and Ccnb2 mRNA and translation of reporters. Oocytes from RiboTag transgenic mice were isolated at the GV stage and matured in vitro for the indicated times. Extracts were then subjected to immunoprecipitation with HA antibody or IgG (RiboTag IP) and the recovery of transcripts in the IP pellet monitored by qPCR. The Mos (A), Ccnb1 (B) and Ccnb2 (C) mRNAs recovery in the IP pellet are reported as solid symbols. Each point is the mean ± SEM, N = 3. In parallel, oocytes from wild type mice were collected and injected with cRNAs coding for the Firefly luciferase and each of the reporters containing Renilla luciferase fused to the Mos, Ccnb1 or Ccnb2 3′UTR. After recovery in media with milrinone, oocytes were released from cell cycle arrest by washing and transferring to media without milrinone. Oocytes were collected at the indicated time points and the accumulation of the luciferase reporter was measured (dashed lines). Each point is the mean ± SEM of at least 5 biological replicates. (D) Accumulation of the endogenous Mos, Cyclin B1 and Cyclin B2 proteins during oocyte maturation was monitored using western blot analysis. Extracts from 100 oocytes per lane were resolved on the SDS PAGE and proteins were detected with specific antibodies, respectively. Tubulin levels were monitored as the loading control. The experiment was repeated twice with identical results.
