Gene expression and protein levels as a function of PEG-3 expression. (A) Northern blotting analysis of steady–state PEG-3, VEGF, Ad5 E1A, and GAPDH RNA in E11 and its variants. (B) PEG-3 and ACTIN protein levels were determined by Western blotting using a peptide-derived PEG-3 polyclonal antibody and an ACTIN mAb, respectively, with total lysates from an equal number of cells. To determine VEGF protein secretion, equal numbers of cells were cultured for 36 hr in medium without serum; the medium was collected and concentrated, and VEGF protein levels were determined by Western blotting using a VEGF mAb and the enhanced chemiluminescence approach. Lane designations: 1: E11-NMT; 2: E11-NMT/PEG-3 AS cl 3; 3: E11; and 4: E11/PEG-3 S cl 13.