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. 2004 Apr 26;165(2):203–211. doi: 10.1083/jcb.200309145

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Copyright © 2004, The Rockefeller University Press

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Figure 4. — Vg1RBP/vera and hnRNP I interact in both the nucleus and cytoplasm. (A) Vg1RBP/vera-FLAG was expressed in stage III/IV oocytes, and nuclear (lanes 1 and 3–6) or cytoplasmic (lanes 2 and 7–10) lysates were prepared. Immunoprecipitations were performed using Sepharose beads plus Vg1RBP/vera S10 lysates (lanes 3 and 7), anti-FLAG beads alone (lanes 4 and 8), anti-FLAG beads with Vg1RBP/vera-FLAG, and nuclear (lanes 5 and 6) or cytoplasmic (lanes 9 and 10) S10 lysate in the presence (lanes 6 and 10) or absence (lanes 5 and 9) of RNase A. Total (lanes 1 and 2) and bound (lanes 3–10) proteins were separated by 10% SDS-PAGE and immunoblotted with anti-hnRNP I. All samples were run on the same gel, but lane order was changed for presentation in the figure, with adjacent lanes boxed together. (B) Nuclear (lanes 1 and 2) and cytoplasmic (lanes 3 and 4) S10 lysates were prepared and were subjected to digestion with RNase A (lanes 2 and 4) or mock digestion (lanes 1 and 3). Vg1 (top) and VegT (bottom) RNAs present in the lysates were detected by RT-PCR. All samples were run on the same gel; adjacent lanes are grouped together. (C) Nuclear (lanes 1 and 3–6) and cytoplasmic (lanes 2 and 7–10) lysates were prepared from oocytes expressing hnRNP I-FLAG. Immunoprecipitations were performed using Sepharose beads plus lysate (lanes 3 and 7), anti-FLAG beads alone (lanes 4 and 8), and anti-FLAG beads and nuclear (lanes 5 and 6) or cytoplasmic (lanes 9 and 10) S10 lysate either with (lanes 6 and 10) or without (lanes 5 and 9) RNase A treatment. Total (lanes 1 and 2) and bound (lanes 3–10) proteins were separated by SDS-PAGE. Bound Vg1RBP/vera was detected by immunoblot with anti-Vg1RBP/vera (top panels), and immunoprecipitated hnRNP I was detected by immunoblot with anti-hnRNP I (bottom panels). All samples were run on the same gel, but lane order was changed for presentation in the figure, with adjacent lanes boxed together.