Fig. 5

E718 restores DLG1 expression despite the presence of E618. a Analysis of total DLG1 expression levels in the presence of HPV-18 E6 and E7 proteins. HEK293 cells were transfected with 1 μg of pmTurq2-DLG1, 5 μg of pgw-HA-E618 and/or 5 μg of pegfp-E718 or pegfp-c1 empty vector (control). Fusion proteins were assessed by western blot using an anti-GFP antibody, which recognizes all fluorescent tagging proteins. γ tubulin levels were used as loading control. b HEK293 cells were transfected with 1 μg of pmTurq2-DLG1, 5 μg of pgw-HA-E618 and 5 μg of pegfp-E718 as indicated. The same amount of DNA was used for each transfection condition, using for balancing the pegfp-c1 empty vector. BTZ proteasome inhibitor was added to the cells 4 h before harvesting, as indicated (+). Fusion proteins was assessed by western blot using an anti-GFP antibody, which recognizes all fluorescent tagging proteins. α tubulin levels were used as loading control. c Changes in DLG1 levels in the soluble and insoluble fraction when E618 and E718 are co-expressed. HEK293 cells co-transfected with 1 μg of mTurq2-DLG1, 5 μg of seyfp2-E618, and/or 5 μg of pegfp-E718 were harvested, and the detergent insoluble and soluble fractions were obtained after centrifugation. Pegfp-c1 empty vector was used as control. Same amounts of protein extracts were resolved by SDS-PAGE and protein expression was ascertained by western blot using anti-GFP. Right, densitometry analysis of DLG1 levels normalized to γ-tubulin expression. Asterisks denote significant difference among conditions tested by one-tailed t-test (*p < 0.05). In a and b, the results shown are representative of three independent experiments. In all cases, cells were harvested after 24 hs post-transfection. β-Gal acted as a control for transfection efficiency in all assays. γ tubulin levels were used as loading control. Full-lenght blots are presented in the Supplementary Figure S5