FIGURE 4.

The uENF1, uENF2, and PP precursor proteins are translated from the uENF1-uENF2-PP tricistronic mRNA in Sf9 cells. A, shown is a schematic representation of mRNAs used in experiments. B, detection of the proPP protein in conditioned medium of Sf9 cells expressing the intact tricistronic mRNA (1-2-PP) by Western blot using an anti-PP antibody is shown. C, detection of V5 epitope-tagged uENF1, uENF2, and proPP proteins in the conditioned medium of Sf9 cells expressing the 1-V5, 1-2-V5, or 1-2-PP-V5 mRNA, respectively, by Western blot using an anti-V5 antibody is shown. To achieve uniform expression levels, a one-fifth volume of the 1-V5 protein solution was loaded on the gel as compared with the others.