FIGURE 1.

Characterization of antibodies and detection of Pum1 and Pum2 in oocytes. A, E. coli-produced Pum1-His (10 ng) (lanes 1, 3, and 5) and Pum2-His (10 ng) (lanes 2, 4, and 6) were probed with anti-His antibody (lanes 1 and 2), anti-Pum1N antibody (lanes 3 and 4), and anti-Pum2N antibody (lanes 5 and 6). B, anti-Pum1N and anti-Pum2N immunoblots of crude extracts from immature (Im) and mature (M) oocytes. C, phosphorylation of Pum1 and Pum2 accompanied by changes in electrophoretic mobility. Extracts from [γ-³²P]ATP-injected immature (Im) and mature (M) oocytes were immunoprecipitated with anti-Pum1 or anti-Pum2 antibody, treated with (+) or without (−) λ-phosphatase (λ-PPase), immunoblotted (IB) with the same antibodies as those used for IP, and subjected to autoradiography (1-month exposure for Pum1 and 1-week exposure for Pum2) to detect the label of ³²P (32P). Extracts before IP were also immunoblotted (Initial). D and E, quantification of the protein contents of endogenous Pum1 (D) and Pum2 (E) in one oocyte. One microliter of immature oocyte extracts (Im), which is equivalent to one oocyte, was immunoblotted with anti-Pum1N and anti-Pum2N antibodies with quantified recombinant Pum1-His and Pum2-His. The signal intensity of individual bands of Pum1 and Pum2 on the immunoblots is expressed as arbitrary units. WB, Western blot.