FIGURE 5.

Interaction of Pumilio with PARN and GLD2. A, oocytes were injected with mRNAs encoding GST-Pum1 or T7-tagged GST and incubated with or without progesterone to induce maturation. Extracts from immature (Im) and mature (M) oocytes were incubated with GSH-Sepharose for GST pulldown assay (GST-PD) or incubated in the presence (+) or absence (−) of anti-PARN antibody for IP (α-PARN-IP). The resulting precipitates were immunoblotted with antibodies (Ab) against Pum1, PARN, and T7 tag (for T7-GST in GST-PD). Extracts before IP were also immunoblotted (Initial). B, similar experiments to those in A using oocytes overexpressing GST-Pum2. C, oocyte extracts used in A were treated with (+) or without (−) RNase A and incubated in the presence (+) or absence (−) of anti-PARN antibody for IP. The resulting precipitates were immunoblotted with antibodies against Pum1 and PARN. Extracts before IP were also immunoblotted (Initial). D, similar experiments to those in C using oocyte extracts used in B. E, oocytes were injected with mRNAs encoding T7-GLD2 and GST-Pum1 and incubated with or without progesterone to induce maturation. Oocytes injected with GST mRNA were used as a control. Extracts from immature (Im) and mature (M) oocytes were subjected to GST pulldown assay (GST-PD) or incubated in the presence (+) or absence (−) of anti-T7 antibody for IP (α-T7-IP). The resulting precipitates were immunoblotted with antibodies against Pum1, T7 tag (for T7-GLD2 and T7-GST in GST-PD), and GLD2 (for T7-GLD2 in α-T7-IP to avoid visualization of the antibody used for IP). F, similar experiments to those in E using oocytes overexpressing GST-Pum2. G, oocyte extracts used in E were treated with (+) or without (−) RNase A and incubated in the presence (+) or absence (−) of anti-T7 antibody for IP. The resulting precipitates were immunoblotted with antibodies against Pum1 and T7 tag (for T7-GLD2). H, similar experiments to those in G using oocyte extracts used in F. I, immature (Im) and mature (M) oocyte extracts were incubated with anti-CPEB (α-CPEB-IP), anti-Pum1N (α-Pum1N-IP), or anti-PARN antibody (α-PARN-IP) or incubated without an antibody (Control IP). Following IP, the samples were subjected to RT-PCR to detect cyclin B1 and β-actin mRNAs. Samples before IP (Initial, RNAs corresponding to 5% of those subjected to IP were used) were also analyzed to confirm the presence of cyclin B1 and β-actin mRNAs. J, oocytes overexpressing T7-GLD2 were treated with or without progesterone to induce maturation. Immature (Im) and mature (M) oocyte extracts were incubated with anti-T7 antibody (α-T7(GLD2)-IP) to precipitate T7-GLD2 or incubated without an antibody as a control (Control IP). Samples were subjected to RT-PCR to detect cyclin B1 and β-actin mRNAs.