Figure 4.

Adenovirus type 2 E1A downregulates EGFR protein and mRNA. (a) Western blot analysis showing expression of EGFR, PARP p85, E1A and actin using F4, G734A, M58 and anti-β-actin primary antibodies, respectively. H357 and Saos-2 cells were infected with Ad-E1A or control Ad-Del adenoviral vectors at a MOI of 10. Cells were lysed 24 and 48 h after infection, equal amounts of total protein from each sample was separated by SDS–PAGE, transferred to nitrocellulose and hybridized to different antibodies as described in the Materials and methods section. (b) E1A-mediated downregulation of EGFR mRNA. H357 cells were either untreated or infected with Ad-Del or Ad-E1A at a MOI of 10 and harvested 48 h after infection. PCR was performed on cDNA generated from whole mRNA. Specific EGFR was amplified using GAPDH as an internal control. The sizes of amplified fragments are indicated. Abbreviations: cDNA, complementary DNA; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MOI, multiplicity of infection; PARP, poly (ADP ribose) polymerase; PCR, polymerase chain reaction; SDS–PAGE, lauryl sulphate–polyacrylamide gel electrophoresis.