Northern blot analysis of total and cytoplasmic GagPol-DrCTE or GagPol-RRE mRNA from transfected 293T cells. (A,B) 293T cells (1 × 107 in a 15-cm culture dish) were transfected with 15 µg pCMVGagPol-DrCTE(2X) (HR4466) and 1 µg pCMV-SEAP (HR1831) with or without plasmids that expressed zebrafish Nxf1 (5 µg) and zebrafish Nxt2 (2.5 µg) proteins. (C,D) 293T cells (1 × 107 in a 15-cm culture dish) were transfected with 15 µg pCMVGagPol-RRE (pHR354) and 1 µg pCMV-SEAP (pHR1831) with or without plasmid pHR30 (5 µg) that expresses the HIV Rev protein. Fifty-five hours post-transfection, total (A,C) and cytoplasmic (B,D) mRNAs were isolated from the transfected cells as described in Materials and Methods. Blots containing mRNAs (5 µg/lane) were hybridized with 32P-labeled GagPol and SEAP probes. Brackets show the fold difference in the levels of the GagPol mRNA between the indicated lanes after normalization for SEAP RNA levels. In C and D the individual lanes ±Rev were put together electronically from different parts of the original gel.
