Fig. 1. A faint amount of Mos protein is synthesized in Mos antisense-injected oocytes. (A) Early process of MAPK monophosphorylation is allowed in Mos antisense-injected oocytes. Timecourse of Mos accumulation and MAPK phosphorylation during maturation of oocytes injected (+ AS Mos) or not (– AS Mos) with Mos antisense oligonucleotides. At the indicated time (in minutes or hours) after progesterone addition, a group of five oocytes were homogenized and analyzed by western blot with the indicated antibodies. The same membrane was used for each antibody after stripping. M indicates that oocytes underwent GVBD. The equivalent of one oocyte was loaded per lane. (B) Only the diphosphorylated form of MAPK is active. Timecourse of MAPK phosphorylation in non injected oocytes was analyzed by western blot using specific antibodies that recognize the monophosphorylated (Ab pY), the diphosphorylated (Ab pTpY) or the total MAPK (Ab ERK). The same samples (equivalent of two oocytes) were analyzed for MAPK activity, using the in gel MBP kinase assay. (C) Quantification of Mos protein levels in Mos antisense-injected oocytes. Successive dilutions of homogenates from non-injected mature oocytes with stage VI oocyte homogenates (to keep the same protein concentration in each sample) were performed to obtain the equivalent of one matured oocyte to 1/20th of a matured oocyte as indicated. These samples were compared with Mos antisense-injected oocytes (equivalent of one oocyte) that underwent (AS Mos M) or not (AS Mos NM) GVBD for the expression of Mos protein. All the oocytes came from the same experiment and the same frog. A control for loading is achieved using ß-tubulin detection.
