Comparison of zebrafish CTE function in 293T cells with added Nxf1 and Nxt from zebrafish and mammals. (A) 293T cells (1.2 × 106) were transfected with 1.8 µg of the CTE-reporter plasmid pCMVGagPol-DrCTE(2X) (HR4466), 90 ng of pCMV-SEAP (HR1931), and plasmids expressing zebrafish or mammalian Nxf1 and Nxt proteins as indicated in the figure. The HsNxf1, DrNxf1, and DrNxt2 proteins contain an amino-terminal Flag-epitope tag. The MmNxt1 protein is not Flag-tagged. DNA transfections, p24 and SEAP activity analyses were performed as described in the legend of Figure 3A. (B) Western blot analysis of protein expression in the transfected 293T cells. Lysates from the transfected cells were separated on denaturing SDS-PAGE gels and analyzed by Western blotting. Blots were probed with an M2 monoclonal anti-Flag antibody (Sigma) and detected with a goat anti-mouse IgG antibody-IRDye 800. Blots were visualized and analyzed using the LI-COR Odyssey infrared imaging system.
