DLG1 is a common target of the E6 proteins, regardless of oncogenic potential.
A. DLG1 binds to the E6 PBMs of both oncogenic and non-oncogenic HPV types; the histogram shows mean peptide numbers from the mass spectroscopy analysis of all pulldowns, analysed using Prism software, error bars show Standard Error of the Mean. B. The non-oncogenic HPV-40 E6 PBM binds DLG1 to a level similar to the HPV-16 E6 PBM. A peptide pulldown from HaCat cell extract, using the control (scr), HPV-16 and HPV-40 peptides, was analysed by western blot and probed for DLG1, CASK, hScrib and ZO-2, as indicated. C. DLG1 binds to the E6 proteins of both oncogenic and non-oncogenic HPV types. In vitro translated radiolabelled DLG1 was incubated with the full-length HPV-66 and HPV-40 E6 proteins expressed as GST fusion proteins. GST alone, GST-HPV-16E6 and HPV-18-E6 were included as controls. The upper panel shows the autoradiograph of bound DLG1 and the lower panel shows the Coomassie stained gel. Arrows indicate the GST-E6 proteins. The histogram shows the quantitation of at least three binding assays, analysed by the ImageJ and Prism programmes. Standard deviations are shown. D. The non-oncogenic HPV-40 E6 protein binds to DLG1 via its PDZ domains. A GST-pulldown assay, using in vitro translated radiolabelled HPV-18 and HPV-40 E6 proteins, together with GST fusion proteins with full-length DLG1(FL), the N-terminus of DLG1, lacking any PDZ domain (NT), and the first 401 amino acid residues of DLG1, containing the PDZ domains 1 and 2 (1/2). The upper panel shows the autoradiograph of the bound E6 proteins, the lower panel shows the Coomassie stained gel. Arrows indicate the GST fusion proteins.