Induction of IL-8 by UL76 is mediated by activation of the DNA Damage response.

<p>A) 293T cells expressing UL76 were lysed 48 h post-transfection and the levels of ATM(S1981), γH2AX, p53(S15) and p53 were detected by western blot. Expression of UL76 was confirmed using an anti-HA-HPR conjugated antibody and detection of tubulin was used as loading control. B) Subcellular localization of NEMO in 293T cells transfected with pcDNA3HA-UL76 or control plasmid was determined by indirect immunofluorescence using an anti-NEMO antibody. UL76 expression was detected using HA-FITC conjugated antibody and DAPI staining was used to define nuclei. Images were acquired with a 100× objective. C) Phosphorylation of NEMO at serine 85 in 293T cells transfected with pcDNA3HA-UL76 or control plasmid was detected at 24 h post-transfection by western blot using a specific anti-p-NEMO(S85) or NEMO antibody. Expression of UL76 was confirmed using an anti-HA-HPR conjugated antibody and detection of β-actin was used as loading control. D) IL-8 concentration in supernatants of 293T cells expressing UL76 or control plasmid in the presence or absence of ATM inhibitor, KU55933 (10 µM), was determined 48 h post-transfection by ELISA. Data are expressed as fold induction to control vector (means ± SD) from one of three similar experiments. Expression of UL76 was confirmed by western blot (below) and detection of β-actin was used as loading control. E) IL-8 concentration in supernatants of <i>ATM -/-</i> cells expressing UL76 or control plasmid was determined 48 h post-transfection by ELISA. Cells were stimulated for 5 h with etoposide (10 µM) or TNFα (10 ng/ml) as controls. Data are expressed as means ± SD of duplicate wells from one of three similar experiments. Expression of UL76 was confirmed by western blot (below) and detection of β-actin was used as loading control.