Fig. 4

Analysis of DLG1 expression in the presence of both E618 and E718 oncoproteins. a Influence of E618 and E718 co-expression on DLG1 localization. The vectors for HA-DLG1 (red), mTurq2-E618 (green) and egfp-E718 (blue) expression were co-transfected in HEK293 cells. Cells were fixed, incubated overnight with an anti-HA antibody and counterstained with Cy3-tagged anti-mouse antibody (upper panel). HEK293 cells were transfected with expression vectors for fluorescence-tagged egfp-DLG1 (red), mTurq2-E6.18 (green) and cherry-E718 (blue) and analysed by confocal microscopy (bottom panel). White arrow indicates DLG1 redistribution. Light-blue arrow indicates DLG1 expression partially overlapped with E618 and E718 expression. Scale bars: 10 μm. The results are representative of at least 6 independent experiments and around 80 triple transfected cells were analysed. PCC was calculated to evaluate E718/DLG1 or E618/DLG1 co-localization (right). The results are representative of at least 6 independent experiments. b Influence of E618 and E718 co-expression on DLG1 localization in A549 cells. Upper panel: the encoding vectors for seyfp2-E618 or cherry-E718 or mTurq2-DLG1 were separately transfected into A549 cells and the localization of each fusion protein was analysed by confocal microscopy (yellow arrows). Bottom panel: A549 cells were transfected with expression vectors for fluorescence-tagged egfp-DLG1 (red), mTurq2-E618 (green) and cherry-E718 (blue) and analysed by confocal microscopy. White arrow indicates DLG1 redistribution (bottom panel). Light blue arrow indicates HA-DLG1 expression partially overlapped with E618 and E718 expression. Scale bars: 10 μm. c E718 does not co-immunoprecipitate with E618/DLG1 protein complexes. HEK293 cells were transfected with pcDNA3-HA-DLG1, pseyfp2-E618 and pegfp-E718 vectors. Equal amounts of protein extracts were immunoprecipitated with anti-HA antibody and bound to Protein A-Sepharose affinity beads. Immunoprecipitates, and a fraction of whole cell extracts (viral protein inputs), were resolved by SDS-PAGE. HA-DLG1 and viral proteins were probed with anti-HA and anti-GFP, respectively, by western blot. Full-lenght blots are presented in the Supplementary Figure S4. The results are representative of at least 3 independent experiments