Figure 2.

Protein synthesis is required for accumulation of ORC6L. (A) ORC6L protein levels were determined during oocyte maturation. Full-grown oocytes (GV) were collected and in vitro matured in the absence (−) or presence of 10 μg/ml cycloheximide (CHX). At the indicated times following removal of milrinone samples were collected for immunoblotting. The immunoblots were performed as described in Fig. 3, using 10 oocytes per lane. The experiment was conducted three times and shown is a representative example. (B) Stability of ORC6L protein in unfertilized eggs. Metaphase II eggs (MII) were collected and incubated in CZB with (CHX) or without (−) cycloheximide, cycoheximide and MG132 (CHX+MG) or MG132 alone (MG). Another group of eggs was parthenogenetically activated with 10 mM SrCl₂ and incubated for 6 h (Sr²⁺). ORC6L protein levels were determined by immunoblotting as described above. The experiment was performed two times and similar results were obtained in each case. A similar experiment without adding MG132 was conducted three times and similar results to those shown here were obtained with respect to the extensive degradation of ORC6L in the presence of cycloheximide. In these experiments, however, the apparent increase in the amount of ORC6L in activated eggs when compared to unactivated eggs was not always observed.