GFP-SseK3 inhibits TNFα stimulated NFκB luciferase activity.

<p><b>(A)</b> Sub-confluent Hela cells were transiently co-transfected with plasmids encoding GFP-SseK3, GFP-SseK3AAA, GFP-NleB1 or GFP-NleB1AAA plasmid together with pNF-κB-Luc plasmid and the control pRL-TK plasmid. 16–18 h post transfection, cells were stimulated with 20 ng/ml of TNFα for 16 h. Following, the cells were harvested and analysed by the dual luciferase reporter assay according to the manufacturer’s instructions using a FluoStar Omega plate reader. The firefly luciferase activity of each sample was normalized to the <i>Renilla</i> luciferase activity. Graph represents the fold increase of NF-κB luciferase activity from three independent experiments. <b>(B)</b> HeLa were transiently transfected with GFP, GFP-SseK3 or GFP-SseK3AAA plasmids. 16–18 h post transfection, cells were stimulated with 20ng/ml of TNFα for 15 and 30 min. Following, the cells were harvested in TK lysis buffer. After BCA protein quantitation, equal amounts of protein samples were subjected for SDS-PAGE and western blots. Membranes were incubated with anti-IκB, anti-phospho Ser536 NFκB p65, or anti-NFκB p65 antibodies, whereas anti-tubulin antibody was included as a loading control. After incubation with IRDye conjugated fluorescence secondary antibodies, fluorescence intensities were detected and scanned by using Li-COR Odyssey infrared imaging system. <b>(C)</b> HeLa non-silence GFP control cells and TRIM32 knockdown cells were transiently transfected with pNF-kB-Luc plasmid, followed by the stimulation with 20 ng/ml of TNFα for 16 h. Cells were harvested for Luciferase reporter assay according to the manufacturer’s instructions. <b>(D)</b> HeLa cells were infected with <i>Salmonella</i> strains for 1 h. After culture medium was replaced by the medium containing 100 μg/ml gentamycin for 1 h, cells were further grown in the medium containing 10 μg/ml gentamycin with untreated or 20 ng/ml TNFα stimulation for additional 6 hrs. The supernatants were collected to measure IL-8 secretion using the Quantikine Human IL-8 Immunoassay according to the manufacturer’s instructions.