FIGURE 6.

(A) uORF peptides were transfected into A549 cells in a time-course experiment. At 6 h or 24 h post-transfection, cells were harvested for Western blot analysis. (17aa) Cells transfected with uORF2 peptide; (17aaC) cells transfected with uORF2 scrambled peptide; (CHX) cells treated with 10 μM cycloheximide; (R) mock transfected group; (N) normal cells. (B) uORF1 and uORF2 peptides were labeled with DyLight Fluor 633 and DyLight Fluor 488, respectively, at the N termini. Fluorescently labeled peptides were transfected into A549 cells in small amounts as tracers. Two hours later, the cells were harvested, fixed, stained with DAPI, and examined with confocal microscopy. (DIC) Differential interference contrast. (C) uORF peptides or scrambled peptides were transfected into A549 cells. Six hours later, cells were harvested and equal amounts of cell lysates were fractionated on an SDS-PAGE gel. Western blot analysis was performed to detect EPHX1 protein expression using a specific monoclonal antibody. β-Actin serves as loading control. Computer densitometry was performed to assess relative quantification of EPHX1 protein expression, normalized to β-actin loading control, and the relative EPHX1 protein levels detected are indicated numerically under the respective lanes of the Western blot results. Each experiment was performed at least two times.