After 18-hour transduction of Ad5 E6/E7 into the A549 cells, the cells were harvested and total proteins were extracted at 4°C with protein lysis buffer consisting of TRIS hydrochloride, 50mM, pH 7.5; sodium chloride, 150mM; EDTA, 5mM; sodium orthovanadate, 2mM; sodium pyrophosphate, 10mM; sodium fluoride, 100mM; glycerol, 10%; Triton X-100, 1%; pepstatin, 10 μg/mL; leupeptin, 20 μg/mL; and aprotinin, 20 μg/mL. Expression of E7, p53, Rb protein, and actin was measured by immunoblotting total cellular protein with the specific E7 (Invitrogen Corp), p53 (Cell Signaling Technology, Inc, Danvers, Massachusetts), pRb (retinoblastoma protein) (BD Pharmingen, San Jose, California), and actin (Santa Cruz Biotechnology, Inc, Santa Cruz, California) antibodies using a standard Western blot technique. After incubation with primary antibodies, proteins were detected with peroxidase-conjugated donkey antigoat or goat antimouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc, West Grove, Pennsylvania) and blots were developed using chemiluminescence (Pierce Biotechnology, Inc, Rockford, Illinois). Jurkat cell lysates (BD Transduction Laboratory, San Jose, California) were used as a positive control for Western blot analysis in cases of Rb protein. Nontransduced cells or cells transduced with A549 were used as controls.