FIG. 8.

Analysis of the Orf22 mutant Δ428. (A) Amino acid composition of Orf22 and spatial arrangement of four of the peptides used in competition assays. (B) GST-Rb was expressed in bacteria and recovered on glutathione-Sepharose; 0, 10, or 50 μg of peptide 395 or 428 was incubated with GST-Rb before addition of lysates of pSG9MOrf22-transfected cells (left and middle) or pSG9ME1A12S-transfected cells (right). The resulting complexes were analyzed by Western blotting with anti-Myc antibody. (C) CEF cells were transfected with 3 μg of Myc-tagged Orf22, Δ428, or E1A 19K construct; 30 h after transfection, cells were lysed and incubated with recombinant GST-Rb. Complex formation was monitored by Western analysis with anti-Myc antibody. (D) A549 cells were cotransfected with 0.12 μg of E2F-Luc reporter construct and 0.16 μg of Orf22 and Δ428 plasmids. Cells were assayed for luciferase activity 24 h after transfection.