Figure 2

Time course expression of pIX-Luc and wild type IX. Western blot analysis of pIX-Luc or pIX expression in cells infected with Ad-wt-pIX-Luc or Ad-wt-E3-Luc. A549 cells were infected with 75 IP/cell of Ad-wt-pIX-Luc, Ad-wt-E3-Luc or uninfected. Cell lysates were collected after 24, 48, and 72 h.p.i. A) 10 μg of protein was boiled in Laemmli sample buffer for 5 minutes and resolved on 4 to 15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane. Staining was performed with an anti-Flag antibody. Lanes 1-3 are lysate from Ad-wt-pIX-Luc-infected cells at 24 hours (Lane 1), 48 hours (Lane 2) or 72 hours (Lane 3). Lanes 4-6 are lysate from Ad-wt-E3-Luc-infected cells at 24 hours (Lane 4), 48 hours (Lane 5) or 72 hours (Lane 6). Lane 7 is lysate analyzed from uninfected cells (unif). The arrow indicates pIX-Luc protein. B) The samples were treated in an identical fashion. Staining was performed with an anti-luciferase antibody. Lanes 1-3 are lysate from Ad-wt-pIX-Luc infected cells at 24 hours (Lane 1), 48 hours (Lane 2) or 72 hours (Lane 3). Lanes 4-6 are lysate from Ad-wt-E3-Luc infected cells at 24 hours (Lane 4), 48 hours (Lane 5) or 72 hours (Lane 6). The solid arrow indicates pIX-Luc protein. The dashed arrow indicates Luc protein. C) The samples were treated in an identical fashion. The staining was performed with anti-pIX. Lane 1 represents uninfected lysate (unif). Lanes 2-4 are lysate from Ad-wt-pIX-Luc-infected cells at 24 hours (Lane 2), 48 hours (Lane 3) or 72 hours (Lane 4). Lanes 5-7 are lysate from Ad-wt-E3-Luc-infected cells at 24 hours (Lane5), 48 hours (Lane 6), or 72 hours (Lane 7). The solid arrow indicates pIX. The dashed arrow indicates truncated pIX-Luc. Signals for Western blots A-C were visualized by diaminobenzidine tablets according to the manufacture's instructions.